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Greenleaf, Arno Lee

Overview:

      My laboratory studies the mechanisms by which different activities in the cell nucleus are connected to the transcription machinery via interactions with the hyper-phosphorylated C-terminal repeat domain (PCTD) of elongating RNA polymerase II. Differential phosphorylation of the CTD, as the RNAP proceeds through successive stages of transcription, orchestrates sequential recruitment of factors to the transcriptase; this serves to coordinate RNA processing events and mRNA nuclear export with gene transcription. To gain a thorough understanding of relevant phosphorylation events on the PCTD, we identified the principal elongation-phase CTD kinase activities in three different eukaryotes, yeast (yCtk1), Drosophila (dCDK12) and humans (hCDK12 & 13).  In addition, we described a novel set of phosphoCTD-associating proteins (“PCAPs”) that we now are investigating primarily in human cells. Our results revealed novel roles for elongating RNAPII, and they engendered several totally new lines of investigation.         


      Recently hCDK12 was shown to be a tumor suppressor for ovarian cancer, and our investigations of this kinase will illuminate its features that, when mutated, can lead to ovarian cancer.


      In another cancer-related project, we are identifying drug targets for a new class of drugs to be aimed at ovarian and breast cancers.

Positions:

Professor of Biochemistry

Biochemistry
School of Medicine

Professor in Molecular Genetics and Microbiology

Molecular Genetics and Microbiology
School of Medicine

Member of the Duke Cancer Institute

Duke Cancer Institute
School of Medicine

Education:

Ph.D. 1974

Ph.D. — Harvard University

Grants:

Phosphorylation and Functions of the RNA Polymerase CTD

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1988
End Date
March 31, 2016

Connections between mRNA elongation and splicing

Administered By
Molecular Genetics and Microbiology
AwardedBy
National Institutes of Health
Role
Co Investigator
Start Date
May 01, 2004
End Date
April 30, 2009

Role of PhosphoCTD in Spliceosome Assembly

Administered By
Biochemistry
AwardedBy
National Science Foundation
Role
Principal Investigator
Start Date
September 01, 2001
End Date
August 31, 2004

Ctd Kinase And Transcription Dynamics

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1996
End Date
June 30, 1999

Ctd Kinase And Rna Polymerase Ii Phosphorylation

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1988
End Date
June 30, 1999

Transcriptase Kinase From Yeast And Drosophila

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1988
End Date
August 31, 1996

Rna Polymerase Ii Subunits And Transcription Factors

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1990
End Date
March 31, 1996

Transcriptase Kinases From Yeast And Drosophila

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
December 01, 1993
End Date
November 30, 1995

Transcriptase Kinase From Drosophila And Yeast

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1990
End Date
November 30, 1991

Transcriptase Kinase From Droxophila And Yeast

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1989
End Date
June 01, 1991

Biochemical Genetics Of Drosophila Rna Polymerase Ii

Administered By
Biochemistry
AwardedBy
National Institutes of Health
Role
Principal Investigator
Start Date
July 01, 1989
End Date
July 01, 1990
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Awards:

Elected Fellow of AAAS. American Association for the Advancement of Science, The.

Type
National
Awarded By
American Association for the Advancement of Science, The
Date
January 01, 2012

Publications:

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors.

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12-cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.

Authors
Zhang, T; Kwiatkowski, N; Olson, CM; Dixon-Clarke, SE; Abraham, BJ; Greifenberg, AK; Ficarro, SB; Elkins, JM; Liang, Y; Hannett, NM; Manz, T; Hao, M; Bartkowiak, B; Greenleaf, AL; Marto, JA; Geyer, M; Bullock, AN; Young, RA; Gray, NS
MLA Citation
Zhang, T, Kwiatkowski, N, Olson, CM, Dixon-Clarke, SE, Abraham, BJ, Greifenberg, AK, Ficarro, SB, Elkins, JM, Liang, Y, Hannett, NM, Manz, T, Hao, M, Bartkowiak, B, Greenleaf, AL, Marto, JA, Geyer, M, Bullock, AN, Young, RA, and Gray, NS. "Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors." Nature chemical biology 12.10 (October 2016): 876-884.
PMID
27571479
Source
epmc
Published In
Nature Chemical Biology
Volume
12
Issue
10
Publish Date
2016
Start Page
876
End Page
884
DOI
10.1038/nchembio.2166

Engineering an analog-sensitive CDK12 cell line using CRISPR/Cas.

The RNA Polymerase II C-terminal domain (CTD) kinase CDK12 has been implicated as a tumor suppressor and regulator of DNA damage response genes. Although much has been learned about CDK12 and its activity, due to the lack of a specific inhibitor and the complications posed by long term RNAi depletion, much is still unknown about the particulars of CDK12 function. Therefore gaining a better understanding of CDK12's roles at the molecular level will be challenging without the development of additional tools. In order to address these issues we have used the CRISPR/Cas gene engineering system to create a mammalian cell line in which the only functional copy of CDK12 is selectively inhibitable by a cell-permeable adenine analog (analog-sensitive CDK12). Inhibition of CDK12 results in a perturbation of the phosphorylation patterns on the CTD and an arrest in cellular proliferation. This cell line should serve as a powerful tool for future studies.

Authors
Bartkowiak, B; Yan, C; Greenleaf, AL
MLA Citation
Bartkowiak, B, Yan, C, and Greenleaf, AL. "Engineering an analog-sensitive CDK12 cell line using CRISPR/Cas." Biochimica et biophysica acta 1849.9 (September 2015): 1179-1187.
PMID
26189575
Source
epmc
Published In
Biochimica et Biophysica Acta: international journal of biochemistry and biophysics
Volume
1849
Issue
9
Publish Date
2015
Start Page
1179
End Page
1187
DOI
10.1016/j.bbagrm.2015.07.010

Expression, purification, and identification of associated proteins of the full-length hCDK12/CyclinK complex.

The coupling of transcription and associated processes has been shown to be dependent on the RNA polymerase II (RNAPII) C-terminal repeat domain (CTD) and the phosphorylation of the heptad repeats of which it is composed (consensus sequence Y1S2P3T4S5P6S7). Two primary S2 position CTD kinases have been identified in higher eukaryotes: P-TEFb and CDK12/CyclinK. The more recently discovered CDK12 appears to act at the 3'-end of the transcription unit and has been identified as a tumor suppressor for ovarian cancer; however much is still unknown about the in vivo roles of CDK12/CyclinK. In an effort to further characterize these roles we have purified to near homogeneity and characterized, full-length, active, human CDK12/CyclinK, and identified hCDK12-associated proteins via mass spectrometry. We find that employing a "2A" peptide-linked multicistronic construct containing CDK12 and CyclinK results in the efficient production of active, recombinant enzyme in the baculovirus/Sf9 expression system. Using GST-CTD fusion protein substrates we find that CDK12/CyclinK prefers a substrate with unmodified repeats or one that mimics prephosphorylation at the S7 position of the CTD; also the enzyme is sensitive to the inhibitor flavopiridol at higher concentrations. Identification of CDK12-associating proteins reveals a strong enrichment for RNA-processing factors suggesting that CDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.

Authors
Bartkowiak, B; Greenleaf, AL
MLA Citation
Bartkowiak, B, and Greenleaf, AL. "Expression, purification, and identification of associated proteins of the full-length hCDK12/CyclinK complex." The Journal of biological chemistry 290.3 (January 2015): 1786-1795.
PMID
25429106
Source
epmc
Published In
The Journal of biological chemistry
Volume
290
Issue
3
Publish Date
2015
Start Page
1786
End Page
1795
DOI
10.1074/jbc.m114.612226

Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat.

The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N-terminal WW domains and the hyperphosphorylated C-terminal domain (CTD) of RNA polymerase II through its C-terminal FF domains. Here, we report biochemical and structural characterization of the C-terminal three FF domains (FF4-6) of TCERG1, revealing a rigid integral domain structure of the tandem FF repeat that interacts with the hyperphosphorylated CTD (PCTD). Although FF4 and FF5 adopt a classical FF domain fold containing three orthogonally packed α helices and a 310 helix, FF6 contains an additional insertion helix between α1 and α2. The formation of the integral tandem FF4-6 repeat is achieved by merging the last helix of the preceding FF domain and the first helix of the following FF domain and by direct interactions between neighboring FF domains. Using peptide column binding assays and NMR titrations, we show that binding of the FF4-6 tandem repeat to the PCTD requires simultaneous phosphorylation at Ser(2), Ser(5), and Ser(7) positions within two consecutive Y(1)S(2)P(3)T(4)S(5)P(6)S(7) heptad repeats. Such a sequence-specific PCTD recognition is achieved through CTD-docking sites on FF4 and FF5 of TCERG1 but not FF6. Our study presents the first example of a nuclear factor requiring all three phospho-Ser marks within the heptad repeat of the CTD for high affinity binding and provides a molecular interpretation for the biochemical connection between the Ser(7) phosphorylation enrichment in the CTD of the transcribing RNA polymerase II over introns and co-transcriptional splicing events.

Authors
Liu, J; Fan, S; Lee, C-J; Greenleaf, AL; Zhou, P
MLA Citation
Liu, J, Fan, S, Lee, C-J, Greenleaf, AL, and Zhou, P. "Specific interaction of the transcription elongation regulator TCERG1 with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5, and Ser7 within the carboxyl-terminal domain repeat." J Biol Chem 288.15 (April 12, 2013): 10890-10901.
PMID
23436654
Source
pubmed
Published In
The Journal of biological chemistry
Volume
288
Issue
15
Publish Date
2013
Start Page
10890
End Page
10901
DOI
10.1074/jbc.M113.460238

Specific interaction of the TCERG1 FF4-6 tandem repeat domains with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5 and Ser7 of the CTD

Authors
Liu, J; Fan, S; Lee, C-J; Greenleaf, AL; Zhou, P
MLA Citation
Liu, J, Fan, S, Lee, C-J, Greenleaf, AL, and Zhou, P. "Specific interaction of the TCERG1 FF4-6 tandem repeat domains with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5 and Ser7 of the CTD." April 2013.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
27
Publish Date
2013

A DNA damage response system associated with the phosphoCTD of elongating RNA polymerase II.

RNA polymerase II translocates across much of the genome and since it can be blocked by many kinds of DNA lesions, detects DNA damage proficiently; it thereby contributes to DNA repair and to normal levels of DNA damage resistance. However, the components and mechanisms that respond to polymerase blockage are largely unknown, except in the case of UV-induced damage that is corrected by nucleotide excision repair. Because elongating RNAPII carries with it numerous proteins that bind to its hyperphosphorylated CTD, we tested for effects of interfering with this binding. We find that expressing a decoy CTD-carrying protein in the nucleus, but not in the cytoplasm, leads to reduced DNA damage resistance. Likewise, inducing aberrant phosphorylation of the CTD, by deleting CTK1, reduces damage resistance and also alters rates of homologous recombination-mediated repair. In line with these results, extant data sets reveal a remarkable, highly significant overlap between phosphoCTD-associating protein genes and DNA damage-resistance genes. For one well-known phosphoCTD-associating protein, the histone methyltransferase Set2, we demonstrate a role in DNA damage resistance, and we show that this role requires the phosphoCTD binding ability of Set2; surprisingly, Set2's role in damage resistance does not depend on its catalytic activity. To explain all of these observations, we posit the existence of a CTD-Associated DNA damage Response (CAR) system, organized around the phosphoCTD of elongating RNAPII and comprising a subset of phosphoCTD-associating proteins.

Authors
Winsor, TS; Bartkowiak, B; Bennett, CB; Greenleaf, AL
MLA Citation
Winsor, TS, Bartkowiak, B, Bennett, CB, and Greenleaf, AL. "A DNA damage response system associated with the phosphoCTD of elongating RNA polymerase II. (Published online)" PLoS One 8.4 (2013): e60909-.
PMID
23613755
Source
pubmed
Published In
PloS one
Volume
8
Issue
4
Publish Date
2013
Start Page
e60909
DOI
10.1371/journal.pone.0060909

Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease.

RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology.

Authors
Möller, A; Xie, SQ; Hosp, F; Lang, B; Phatnani, HP; James, S; Ramirez, F; Collin, GB; Naggert, JK; Babu, MM; Greenleaf, AL; Selbach, M; Pombo, A
MLA Citation
Möller, A, Xie, SQ, Hosp, F, Lang, B, Phatnani, HP, James, S, Ramirez, F, Collin, GB, Naggert, JK, Babu, MM, Greenleaf, AL, Selbach, M, and Pombo, A. "Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease." Molecular & cellular proteomics : MCP 11.6 (2012): M111.011767-.
PMID
22199231
Source
scival
Published In
Molecular & cellular proteomics : MCP
Volume
11
Issue
6
Publish Date
2012
Start Page
M111.011767
DOI
10.1074/mcp.M111.011767

Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease

RNA polymerase II (RNAPII) transcribes protein-coding genes in eukaryotes and interacts with factors involved in chromatin remodeling, transcriptional activation, elongation, and RNA processing. Here, we present the isolation of native RNAPII complexes using mild extraction conditions and immunoaffinity purification. RNAPII complexes were extracted from mitotic cells, where they exist dissociated from chromatin. The proteomic content of native complexes in total and size-fractionated extracts was determined using highly sensitive LC-MS/MS. Protein associations with RNAPII were validated by high-resolution immunolocalization experiments in both mitotic cells and in interphase nuclei. Functional assays of transcriptional activity were performed after siRNA-mediated knockdown. We identify >400 RNAPII associated proteins in mitosis, among these previously uncharacterized proteins for which we show roles in transcriptional elongation. We also identify, as novel functional RNAPII interactors, two proteins involved in human disease, ALMS1 and TFG, emphasizing the importance of gene regulation for normal development and physiology. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Authors
Möller, A; Xie, SQ; Hosp, F; Lang, B; Phatnani, HP; James, S; Ramirez, F; Collin, GB; Naggert, JK; Babu, MM; Greenleaf, AL; Selbach, M; Pombo, A
MLA Citation
Möller, A, Xie, SQ, Hosp, F, Lang, B, Phatnani, HP, James, S, Ramirez, F, Collin, GB, Naggert, JK, Babu, MM, Greenleaf, AL, Selbach, M, and Pombo, A. "Proteomic analysis of mitotic RNA polymerase II reveals novel interactors and association with proteins dysfunctional in disease." Molecular and Cellular Proteomics 11.6 (2012).
Source
scival
Published In
Molecular & cellular proteomics : MCP
Volume
11
Issue
6
Publish Date
2012
DOI
10.1074/mcp.M111.011767

Cotranscriptional association of mRNA export factor Yra1 with C-terminal domain of RNA polymerase II.

The unique C-terminal domain (CTD) of RNA polymerase II, composed of tandem heptad repeats of the consensus sequence YSPTSPS, is subject to differential phosphorylation throughout the transcription cycle. Several RNA processing factors have been shown to bind the phosphorylated CTD and use it to localize to nascent pre-mRNA during transcription. In Saccharomyces cerevisiae, the mRNA export protein Yra1 (ALY/RNA export factor in metazoa) cotranscriptionally associates with mRNA and delivers it to the nuclear pore complex for export to the cytoplasm. Here we report that Yra1 directly binds in vitro the hyperphosphorylated form of the CTD characteristic of elongating RNA polymerase II and contains a phospho-CTD-interacting domain within amino acids 18-184, which also include an "RNA recognition motif" (RRM) (residues 77-184). Using UV cross-linking, we showed that the RRM alone binds RNA, although a larger segment extending to the C terminus (amino acids 77-226) displayed stronger RNA binding activity. Although the RRM is implicated in both RNA and CTD binding, RRM point mutations separated these two functions. Both functions are important in vivo as RNA binding-defective or CTD binding-defective versions of Yra1 engendered growth and mRNA export defects. We also report the construction and characterization of a useful new temperature-sensitive YRA1 allele (R107A/F126A). Using ChIP, we demonstrated that removing the N-terminal 76 amino acids of Yra1 (all of the phospho-CTD-interacting domain up to the RRM) results in a 10-fold decrease in Yra1 recruitment to genes during elongation. These results indicate that the phospho-CTD is likely involved directly in the cotranscriptional recruitment of Yra1.

Authors
MacKellar, AL; Greenleaf, AL
MLA Citation
MacKellar, AL, and Greenleaf, AL. "Cotranscriptional association of mRNA export factor Yra1 with C-terminal domain of RNA polymerase II." J Biol Chem 286.42 (October 21, 2011): 36385-36395.
PMID
21856751
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
42
Publish Date
2011
Start Page
36385
End Page
36395
DOI
10.1074/jbc.M111.268144

Phosphorylation of RNAPII: To P-TEFb or not to P-TEFb?

The C-terminal domain of RNA polymerase II undergoes a cycle of phosphorylation which allows it to temporally couple transcription with transcription-associated processes. The characterization of hitherto unrecognized metazoan elongation phase CTD kinase activities expands our understanding of this coupling. We discuss the circumstances that delayed the recognition of these kinase activities.

Authors
Bartkowiak, B; Greenleaf, AL
MLA Citation
Bartkowiak, B, and Greenleaf, AL. "Phosphorylation of RNAPII: To P-TEFb or not to P-TEFb?." Transcription 2.3 (May 2011): 115-119.
PMID
21826281
Source
pubmed
Published In
Transcription
Volume
2
Issue
3
Publish Date
2011
Start Page
115
End Page
119
DOI
10.4161/trns.2.3.15004

Association of mRNA export factor Yra1 with the C-terminal Domain of RNA Polymerase II: a mechanism for cotranscriptional recruitment

Authors
MacKellar, AL; Greenleaf, AL
MLA Citation
MacKellar, AL, and Greenleaf, AL. "Association of mRNA export factor Yra1 with the C-terminal Domain of RNA Polymerase II: a mechanism for cotranscriptional recruitment." April 2011.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
25
Publish Date
2011

cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72.

RNA polymerase II coordinates co-transcriptional events by recruiting distinct sets of nuclear factors to specific stages of transcription via changes of phosphorylation patterns along its C-terminal domain (CTD). Although it has become increasingly clear that proline isomerization also helps regulate CTD-associated processes, the molecular basis of its role is unknown. Here, we report the structure of the Ser(P)(5) CTD phosphatase Ssu72 in complex with substrate, revealing a remarkable CTD conformation with the Ser(P)(5)-Pro(6) motif in the cis configuration. We show that the cis-Ser(P)(5)-Pro(6) isomer is the minor population in solution and that Ess1-catalyzed cis-trans-proline isomerization facilitates rapid dephosphorylation by Ssu72, providing an explanation for recently discovered in vivo connections between these enzymes and a revised model for CTD-mediated small nuclear RNA termination. This work presents the first structural evidence of a cis-proline-specific enzyme and an unexpected mechanism of isomer-based regulation of phosphorylation, with broad implications for CTD biology.

Authors
Werner-Allen, JW; Lee, C-J; Liu, P; Nicely, NI; Wang, S; Greenleaf, AL; Zhou, P
MLA Citation
Werner-Allen, JW, Lee, C-J, Liu, P, Nicely, NI, Wang, S, Greenleaf, AL, and Zhou, P. "cis-Proline-mediated Ser(P)5 dephosphorylation by the RNA polymerase II C-terminal domain phosphatase Ssu72." J Biol Chem 286.7 (February 18, 2011): 5717-5726.
PMID
21159777
Source
pubmed
Published In
The Journal of biological chemistry
Volume
286
Issue
7
Publish Date
2011
Start Page
5717
End Page
5726
DOI
10.1074/jbc.M110.197129

Updating the CTD Story: From Tail to Epic.

Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA but also coordinates transcription-related processes via its unique C-terminal repeat domain (CTD). The CTD is an RNAPII-specific protein segment consisting of repeating heptads with the consensus sequence Y(1)S(2)P(3)T(4)S(5)P(6)S(7) that has been shown to be extensively post-transcriptionally modified in a coordinated, but complicated, manner. Recent discoveries of new modifications, kinases, and binding proteins have challenged previously established paradigms. In this paper, we examine results and implications of recent studies related to modifications of the CTD and the respective enzymes; we also survey characterizations of new CTD-binding proteins and their associated processes and new information regarding known CTD-binding proteins. Finally, we bring into focus new results that identify two additional CTD-associated processes: nucleocytoplasmic transport of mRNA and DNA damage and repair.

Authors
Bartkowiak, B; Mackellar, AL; Greenleaf, AL
MLA Citation
Bartkowiak, B, Mackellar, AL, and Greenleaf, AL. "Updating the CTD Story: From Tail to Epic." Genet Res Int 2011 (2011): 623718-.
PMID
22567360
Source
pubmed
Published In
Genetics Research International
Volume
2011
Publish Date
2011
Start Page
623718
DOI
10.4061/2011/623718

CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1.

Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 5' end and higher in the middle and at the 3' end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1(ts) mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 ↔ CDK9 and Ctk1 ↔ CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1.

Authors
Bartkowiak, B; Liu, P; Phatnani, HP; Fuda, NJ; Cooper, JJ; Price, DH; Adelman, K; Lis, JT; Greenleaf, AL
MLA Citation
Bartkowiak, B, Liu, P, Phatnani, HP, Fuda, NJ, Cooper, JJ, Price, DH, Adelman, K, Lis, JT, and Greenleaf, AL. "CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1." Genes Dev 24.20 (October 15, 2010): 2303-2316.
PMID
20952539
Source
pubmed
Published In
Genes & development
Volume
24
Issue
20
Publish Date
2010
Start Page
2303
End Page
2316
DOI
10.1101/gad.1968210

The phosphoCTD-interacting domain of Topoisomerase I.

The N-terminal domain (NTD) of Drosophila melanogaster (Dm) Topoisomerase I has been shown to bind to RNA polymerase II, but the domain of RNAPII with which it interacts is not known. Using bacterially-expressed fusion proteins carrying all or half of the NTDs of Dm and human (Homo sapiens, Hs) Topo I, we demonstrate that the N-terminal half of each NTD binds directly to the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of the largest RNAPII subunit, Rpb1. Thus, the amino terminal segment of metazoan Topo I (1-157 for Dm and 1-114 for Hs) contains a novel phosphoCTD-interacting domain that we designate the Topo I-Rpb1 interacting (TRI) domain. The long-known in vivo association of Topo I with active genes presumably can be attributed, wholly or in part, to the TRI domain-mediated binding of Topo I to the phosphoCTD of transcribing RNAPII.

Authors
Wu, J; Phatnani, HP; Hsieh, T-S; Greenleaf, AL
MLA Citation
Wu, J, Phatnani, HP, Hsieh, T-S, and Greenleaf, AL. "The phosphoCTD-interacting domain of Topoisomerase I." Biochem Biophys Res Commun 397.1 (June 18, 2010): 117-119.
PMID
20493173
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
397
Issue
1
Publish Date
2010
Start Page
117
End Page
119
DOI
10.1016/j.bbrc.2010.05.081

RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription

It is known that transcription can induce DNA recombination, thus compromising genomic stability. RECQ5 DNA helicase promotes genomic stability by regulating homologous recombination. Recent studies have shown that RECQ5 forms a stable complex with RNA polymerase II (RNAPII) in human cells, but the cellular role of this association is not understood. Here, we provide evidence that RECQ5 specifically binds to the Ser2,5-phosphorylated C-terminal repeat domain (CTD) of the largest subunit of RNAPII, RPB1, by means of a Set2-Rpb1-interacting (SRI) motif located at the C-terminus of RECQ5. We also show that RECQ5 associates with RNAPII-transcribed genes in a manner dependent on the SRI motif. Notably, RECQ5 density on transcribed genes correlates with the density of Ser2-CTD phosphorylation, which is associated with the productive elongation phase of transcription. Furthermore, we show that RECQ5 negatively affects cell viability upon inhibition of spliceosome assembly, which can lead to the formation of mutagenic R-loop structures. These data indicate that RECQ5 binds to the elongating RNAPII complex and support the idea that RECQ5 plays a role in the maintenance of genomic stability during transcription. © 2010 The Author(s).

Authors
Kanagaraj, R; Huehn, D; MacKellar, A; Menigatti, M; Zheng, L; Urban, V; Shevelev, I; Greenleaf, AL; Janscak, P
MLA Citation
Kanagaraj, R, Huehn, D, MacKellar, A, Menigatti, M, Zheng, L, Urban, V, Shevelev, I, Greenleaf, AL, and Janscak, P. "RECQ5 helicase associates with the C-terminal repeat domain of RNA polymerase II during productive elongation phase of transcription." Nucleic Acids Research 38.22 (2010): 8131-8140.
PMID
20705653
Source
scival
Published In
Nucleic Acids Research
Volume
38
Issue
22
Publish Date
2010
Start Page
8131
End Page
8140
DOI
10.1093/nar/gkq697

Genetic organization, length conservation, and evolution of RNA polymerase II carboxyl-terminal domain

With a simple tandem iterated sequence, the carboxyl terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II) serves as the central coordinator of mRNA synthesis by harmonizing a diversity of sequential interactions with transcription and processing factors. Despite intense research interest, many key questions regarding functional and evolutionary constraints on the CTD remain unanswered; for example, what selects for the canonical heptad sequence, its tandem array across organismal diversity, and constant CTD length within given species and finally and how a sequence-identical, repetitive structure can orchestrate a diversity of simultaneous and sequential, stage-dependent interactions with both modifying enzymes and binding partners? Here we examine comparative sequence evolution of 58 RNAP II CTDs from diverse taxa representing all six major eukaryotic supergroups and employ integrated evolutionary genetic, biochemical, and biophysical analyses of the yeast CTD to further clarify how this repetitive sequence must be organized for optimal RNAP II function. We find that the CTD is composed of indivisible and independent functional units that span diheptapeptides and not only a flexible conformation around each unit but also an elastic overall structure is required. More remarkably, optimal CTD function always is achieved at approximately wild-type CTD length rather than number of functional units, regardless of the characteristics of the sequence present. Our combined observations lead us to advance an updated CTD working model, in which functional, and therefore, evolutionary constraints require a flexible CTD conformation determined by the CTD sequence and tandem register to accommodate the diversity of CTD-protein interactions and a specific CTD length rather than number of functional units to correctly order and organize global CTD-protein interactions. Patterns of conservation of these features across evolutionary diversity have important implications for comparative RNAP II function in eukaryotes and can more clearly direct specific research on CTD function in currently understudied organisms. © 2010 The Author.

Authors
Liu, P; Kenney, JM; Stiller, JW; Greenleaf, AL
MLA Citation
Liu, P, Kenney, JM, Stiller, JW, and Greenleaf, AL. "Genetic organization, length conservation, and evolution of RNA polymerase II carboxyl-terminal domain." Molecular Biology and Evolution 27.11 (2010): 2628-2641.
PMID
20558594
Source
scival
Published In
Molecular Biology and Evolution
Volume
27
Issue
11
Publish Date
2010
Start Page
2628
End Page
2641
DOI
10.1093/molbev/msq151

Comparative genome-wide screening identifies a conserved doxorubicin repair network that is diploid specific in Saccharomyces cerevisiae.

The chemotherapeutic doxorubicin (DOX) induces DNA double-strand break (DSB) damage. In order to identify conserved genes that mediate DOX resistance, we screened the Saccharomyces cerevisiae diploid deletion collection and identified 376 deletion strains in which exposure to DOX was lethal or severely reduced growth fitness. This diploid screen identified 5-fold more DOX resistance genes than a comparable screen using the isogenic haploid derivative. Since DSB damage is repaired primarily by homologous recombination in yeast, and haploid cells lack an available DNA homolog in G1 and early S phase, this suggests that our diploid screen may have detected the loss of repair functions in G1 or early S phase prior to complete DNA replication. To test this, we compared the relative DOX sensitivity of 30 diploid deletion mutants identified under our screening conditions to their isogenic haploid counterpart, most of which (n = 26) were not detected in the haploid screen. For six mutants (bem1Delta, ctf4Delta, ctk1Delta, hfi1Delta,nup133Delta, tho2Delta) DOX-induced lethality was absent or greatly reduced in the haploid as compared to the isogenic diploid derivative. Moreover, unlike WT, all six diploid mutants displayed severe G1/S phase cell cycle progression defects when exposed to DOX and some were significantly enhanced (ctk1Delta and hfi1Delta) or deficient (tho2Delta) for recombination. Using these and other "THO2-like" hypo-recombinogenic, diploid-specific DOX sensitive mutants (mft1Delta, thp1Delta, thp2Delta) we utilized known genetic/proteomic interactions to construct an interactive functional genomic network which predicted additional DOX resistance genes not detected in the primary screen. Most (76%) of the DOX resistance genes detected in this diploid yeast screen are evolutionarily conserved suggesting the human orthologs are candidates for mediating DOX resistance by impacting on checkpoint and recombination functions in G1 and/or early S phases.

Authors
Westmoreland, TJ; Wickramasekara, SM; Guo, AY; Selim, AL; Winsor, TS; Greenleaf, AL; Blackwell, KL; Olson, JA; Marks, JR; Bennett, CB
MLA Citation
Westmoreland, TJ, Wickramasekara, SM, Guo, AY, Selim, AL, Winsor, TS, Greenleaf, AL, Blackwell, KL, Olson, JA, Marks, JR, and Bennett, CB. "Comparative genome-wide screening identifies a conserved doxorubicin repair network that is diploid specific in Saccharomyces cerevisiae. (Published online)" PLoS One 4.6 (June 8, 2009): e5830-.
PMID
19503795
Source
pubmed
Published In
PloS one
Volume
4
Issue
6
Publish Date
2009
Start Page
e5830
DOI
10.1371/journal.pone.0005830

Association of mRNA Export Factor Yra1 with the C-terminal Domain of RNA Polymerase II

Authors
MacKellar, AL; Greenleaf, AL
MLA Citation
MacKellar, AL, and Greenleaf, AL. "Association of mRNA Export Factor Yra1 with the C-terminal Domain of RNA Polymerase II." FASEB JOURNAL 23 (April 2009).
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
23
Publish Date
2009

Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction.

BRCA1 has been implicated in numerous DNA repair pathways that maintain genome integrity, however the function responsible for its tumor suppressor activity in breast cancer remains obscure. To identify the most highly conserved of the many BRCA1 functions, we screened the evolutionarily distant eukaryote Saccharomyces cerevisiae for mutants that suppressed the G1 checkpoint arrest and lethality induced following heterologous BRCA1 expression. A genome-wide screen in the diploid deletion collection combined with a screen of ionizing radiation sensitive gene deletions identified mutants that permit growth in the presence of BRCA1. These genes delineate a metabolic mRNA pathway that temporally links transcription elongation (SPT4, SPT5, CTK1, DEF1) to nucleopore-mediated mRNA export (ASM4, MLP1, MLP2, NUP2, NUP53, NUP120, NUP133, NUP170, NUP188, POM34) and cytoplasmic mRNA decay at P-bodies (CCR4, DHH1). Strikingly, BRCA1 interacted with the phosphorylated RNA polymerase II (RNAPII) carboxy terminal domain (P-CTD), phosphorylated in the pattern specified by the CTDK-I kinase, to induce DEF1-dependent cleavage and accumulation of a RNAPII fragment containing the P-CTD. Significantly, breast cancer associated BRCT domain defects in BRCA1 that suppressed P-CTD cleavage and lethality in yeast also suppressed the physical interaction of BRCA1 with human SPT5 in breast epithelial cells, thus confirming SPT5 as a relevant target of BRCA1 interaction. Furthermore, enhanced P-CTD cleavage was observed in both yeast and human breast cells following UV-irradiation indicating a conserved eukaryotic damage response. Moreover, P-CTD cleavage in breast epithelial cells was BRCA1-dependent since damage-induced P-CTD cleavage was only observed in the mutant BRCA1 cell line HCC1937 following ectopic expression of wild type BRCA1. Finally, BRCA1, SPT5 and hyperphosphorylated RPB1 form a complex that was rapidly degraded following MMS treatment in wild type but not BRCA1 mutant breast cells. These results extend the mechanistic links between BRCA1 and transcriptional consequences in response to DNA damage and suggest an important role for RNAPII P-CTD cleavage in BRCA1-mediated cancer suppression.

Authors
Bennett, CB; Westmoreland, TJ; Verrier, CS; Blanchette, CAB; Sabin, TL; Phatnani, HP; Mishina, YV; Huper, G; Selim, AL; Madison, ER; Bailey, DD; Falae, AI; Galli, A; Olson, JA; Greenleaf, AL; Marks, JR
MLA Citation
Bennett, CB, Westmoreland, TJ, Verrier, CS, Blanchette, CAB, Sabin, TL, Phatnani, HP, Mishina, YV, Huper, G, Selim, AL, Madison, ER, Bailey, DD, Falae, AI, Galli, A, Olson, JA, Greenleaf, AL, and Marks, JR. "Yeast screens identify the RNA polymerase II CTD and SPT5 as relevant targets of BRCA1 interaction. (Published online)" PLoS One 3.1 (January 16, 2008): e1448-.
Website
http://hdl.handle.net/10161/4482
PMID
18197258
Source
pubmed
Published In
PloS one
Volume
3
Issue
1
Publish Date
2008
Start Page
e1448
DOI
10.1371/journal.pone.0001448

The essential sequence elements required for RNAP II carboxyl-terminal domain function in yeast and their evolutionary conservation

The carboxyl-terminal domain (CTD) of eukaryotic RNA polymerase II is the staging platform for numerous proteins involved in transcription initiation, mRNA processing, and general coordination of nuclear events. Concordant with these central roles in cellular metabolism, the consensus sequence, tandemly repeated structure, and core functions of the CTD are conserved across diverse eukaryotic lineages; however, in other eukaryotes, the CTD has been allowed to degenerate completely. Even in groups where the CTD is strongly conserved, genetic analyses and comparative genomic investigations show that a variety of individual substitutions and insertions are permissible. Therefore, the specific functional constraints reflected by the CTD's conservation across much of eukaryotic evolution have remained somewhat puzzling. Here we propose a hypothesis to explain that strong conservation in budding yeast, based on both comparative and experimental evidence. Through genetic complementation for CTD function, we identify 2 sequence elements contained within pairs of heptapeptides, "Y1-Y8" and "S 2-S5-S9," which are required for all essential CTD functions in yeast. The dual requirements of these motifs can account for strong purifying selection on the canonical CTD heptapeptide. Further, in vitro analysis of GST-CTD fusion proteins as substrates for multiple CTD-directed kinases show reduced phosphorylation efficiencies with increased distance between functional units. This indicates that requirements of the RNAP II phosphorylation cycle are most likely responsible for the strong purifying selection on tandemly repeated CTD structure. © The Author 2008. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

Authors
Liu, P; Greenleaf, AL; Stiller, JW
MLA Citation
Liu, P, Greenleaf, AL, and Stiller, JW. "The essential sequence elements required for RNAP II carboxyl-terminal domain function in yeast and their evolutionary conservation." Molecular Biology and Evolution 25.4 (2008): 719-727.
PMID
18209193
Source
scival
Published In
Molecular Biology and Evolution
Volume
25
Issue
4
Publish Date
2008
Start Page
719
End Page
727
DOI
10.1093/molbev/msn017

Phosphorylation and functions of the RNA polymerase II CTD.

The C-terminal repeat domain (CTD), an unusual extension appended to the C terminus of the largest subunit of RNA polymerase II, serves as a flexible binding scaffold for numerous nuclear factors; which factors bind is determined by the phosphorylation patterns on the CTD repeats. Changes in phosphorylation patterns, as polymerase transcribes a gene, are thought to orchestrate the association of different sets of factors with the transcriptase and strongly influence functional organization of the nucleus. In this review we appraise what is known, and what is not known, about patterns of phosphorylation on the CTD of RNA polymerases II at the beginning, the middle, and the end of genes; the proposal that doubly phosphorylated repeats are present on elongating polymerase is explored. We discuss briefly proteins known to associate with the phosphorylated CTD at the beginning and ends of genes; we explore in more detail proteins that are recruited to the body of genes, the diversity of their functions, and the potential consequences of tethering these functions to elongating RNA polymerase II. We also discuss accumulating structural information on phosphoCTD-binding proteins and how it illustrates the variety of binding domains and interaction modes, emphasizing the structural flexibility of the CTD. We end with a number of open questions that highlight the extent of what remains to be learned about the phosphorylation and functions of the CTD.

Authors
Phatnani, HP; Greenleaf, AL
MLA Citation
Phatnani, HP, and Greenleaf, AL. "Phosphorylation and functions of the RNA polymerase II CTD." Genes Dev 20.21 (November 1, 2006): 2922-2936. (Review)
PMID
17079683
Source
pubmed
Published In
Genes & development
Volume
20
Issue
21
Publish Date
2006
Start Page
2922
End Page
2936
DOI
10.1101/gad.1477006

Structure and CTD phosphorylation pattern-binding specificity of the SRI domain of Set2

Authors
Greenleaf, AL; Phatnani, HP; Li, M; Zhou, P
MLA Citation
Greenleaf, AL, Phatnani, HP, Li, M, and Zhou, P. "Structure and CTD phosphorylation pattern-binding specificity of the SRI domain of Set2." March 6, 2006.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
20
Issue
4
Publish Date
2006
Start Page
A467
End Page
A467

NMR assignment of the SRI domain of human Set2/HYPB.

Authors
Li, M; Phatnani, HP; Greenleaf, AL; Zhou, P
MLA Citation
Li, M, Phatnani, HP, Greenleaf, AL, and Zhou, P. "NMR assignment of the SRI domain of human Set2/HYPB." J Biomol NMR 36 Suppl 1 (2006): 5-. (Letter)
PMID
16435090
Source
pubmed
Published In
Journal of Biomolecular NMR
Volume
36 Suppl 1
Publish Date
2006
Start Page
5
DOI
10.1007/s10858-005-4690-8

Solution structure of the Set2-Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1.

The phosphorylation state of the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II changes as polymerase transcribes a gene, and the distinct forms of the phospho-CTD (PCTD) recruit different nuclear factors to elongating polymerase. The Set2 histone methyltransferase from yeast was recently shown to bind the PCTD of elongating RNA polymerase II by means of a novel domain termed the Set2-Rpb1 interacting (SRI) domain. Here, we report the solution structure of the SRI domain in human Set2 (hSRI domain), which adopts a left-turned three-helix bundle distinctly different from other structurally characterized PCTD-interacting domains. NMR titration experiments mapped the binding surface of the hSRI domain to helices 1 and 2, and Biacore binding studies showed that the domain binds preferably to [Ser-2 + Ser-5]-phosphorylated CTD peptides containing two or more heptad repeats. Point-mutagenesis studies identified five residues critical for PCTD binding. In view of the differential effects of these point mutations on binding to different CTD phosphopeptides, we propose a model for the hSRI domain interaction with the PCTD.

Authors
Li, M; Phatnani, HP; Guan, Z; Sage, H; Greenleaf, AL; Zhou, P
MLA Citation
Li, M, Phatnani, HP, Guan, Z, Sage, H, Greenleaf, AL, and Zhou, P. "Solution structure of the Set2-Rpb1 interacting domain of human Set2 and its interaction with the hyperphosphorylated C-terminal domain of Rpb1." Proc Natl Acad Sci U S A 102.49 (December 6, 2005): 17636-17641.
PMID
16314571
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
102
Issue
49
Publish Date
2005
Start Page
17636
End Page
17641
DOI
10.1073/pnas.0506350102

A novel domain in Set2 mediates RNA polymerase II interaction and couples histone H3 K36 methylation with transcript elongation

Histone methylation and the enzymes that mediate it are important regulators of chromatin structure and gene transcription. In particular, the histone H3 lysine 36 (K36) methyltransferase Set2 has recently been shown to associate with the phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAPII), implying that this enzyme has an important role in the transcription elongation process. Here we show that a novel domain in the C terminus of Set2 is responsible for interaction between Set2 and RNAPII. This domain, termed the Set2 Rpb1 interacting (SRI) domain, is encompassed by amino acid residues 619 to 718 in Set2 and is found to occur in a number of putative Set2 homologs from Schizosaccharomyces pombe to humans. Unexpectedly, BIACORE analysis reveals that the SRI domain binds specifically, and with high affinity, to CTD repeats that are doubly modified (serine 2 and serine 5 phosphorylated), indicating that Set2 association across the body of genes requires a specific pattern of phosphorylated RNAPII. Deletion of the SRI domain not only abolishes Set2-RNAPII interaction but also abolishes K36 methylation in vivo, indicating that this interaction is required for establishing K36 methylation on chromatin. Using 6-azauracil (6AU) as an indicator of transcription elongation defects, we found that deletion of the SRI domain conferred a strong resistance to this compound, which was identical to that observed with set2 deletion mutants. Furthermore, yeast strains carrying set2 alleles that are catalytically inactive or yeast strains bearing point mutations at K36 were also found to be resistant to 6AU. These data suggest that it is the methylation by Set2 that affects transcription elongation. In agreement with this, we have determined that deletion of SET2, its SRI domain, or amino acid substitutions at K36 result in an alteration of RNAPII occupancy levels over transcribing genes. Taken together, these data indicate K36 methylation, established by the SRI domain-mediated association of Set2 with RNAPII, plays an important role in the transcription elongation process. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Authors
Kizer, KO; Phatnani, HP; Shibata, Y; Hall, H; Greenleaf, AL; Strahl, BD
MLA Citation
Kizer, KO, Phatnani, HP, Shibata, Y, Hall, H, Greenleaf, AL, and Strahl, BD. "A novel domain in Set2 mediates RNA polymerase II interaction and couples histone H3 K36 methylation with transcript elongation." Molecular and Cellular Biology 25.8 (2005): 3305-3316.
PMID
15798214
Source
scival
Published In
Molecular and Cellular Biology
Volume
25
Issue
8
Publish Date
2005
Start Page
3305
End Page
3316
DOI
10.1128/MCB.25.8.3305-3316.2005

Expanding the functional repertoire of CTD kinase I and RNA polymerase II: novel phosphoCTD-associating proteins in the yeast proteome.

CTD kinase I (CTDK-I) of Saccharomyces cerevisiae is required for normal phosphorylation of the C-terminal repeat domain (CTD) on elongating RNA polymerase II. To elucidate cellular roles played by this kinase and the hyperphosphorylated CTD (phosphoCTD) it generates, we systematically searched yeast extracts for proteins that bound to the phosphoCTD made by CTDK-I in vitro. Initially, using a combination of far-western blotting and phosphoCTD affinity chromatography, we discovered a set of novel phosphoCTD-associating proteins (PCAPs) implicated in a variety of nuclear functions. We identified the phosphoCTD-interacting domains of a number of these PCAPs, and in several test cases (namely, Set2, Ssd1, and Hrr25) adduced evidence that phosphoCTD binding is functionally important in vivo. Employing surface plasmon resonance (BIACORE) analysis, we found that recombinant versions of these and other PCAPs bind preferentially to CTD repeat peptides carrying SerPO(4) residues at positions 2 and 5 of each seven amino acid repeat, consistent with the positional specificity of CTDK-I in vitro [Jones, J. C., et al. (2004) J. Biol. Chem. 279, 24957-24964]. Subsequently, we used a synthetic CTD peptide with three doubly phosphorylated repeats (2,5P) as an affinity matrix, greatly expanding our search for PCAPs. This resulted in identification of approximately 100 PCAPs and associated proteins representing a wide range of functions (e.g., transcription, RNA processing, chromatin structure, DNA metabolism, protein synthesis and turnover, RNA degradation, snRNA modification, and snoRNP biogenesis). The varied nature of these PCAPs and associated proteins points to an unexpectedly diverse set of connections between Pol II elongation and other processes, conceptually expanding the role played by CTD phosphorylation in functional organization of the nucleus.

Authors
Phatnani, HP; Jones, JC; Greenleaf, AL
MLA Citation
Phatnani, HP, Jones, JC, and Greenleaf, AL. "Expanding the functional repertoire of CTD kinase I and RNA polymerase II: novel phosphoCTD-associating proteins in the yeast proteome." Biochemistry 43.50 (December 21, 2004): 15702-15719.
PMID
15595826
Source
pubmed
Published In
Biochemistry
Volume
43
Issue
50
Publish Date
2004
Start Page
15702
End Page
15719
DOI
10.1021/bi048364h

C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats.

The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is composed of tandem heptad repeats with consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. In yeast, this heptad sequence is repeated about 26 times, and it becomes hyperphosphorylated during transcription predominantly at serines 2 and 5. A network of kinases and phosphatases combine to determine the CTD phosphorylation pattern. We sought to determine the positional specificity of phosphorylation by yeast CTD kinase-I (CTDK-I), an enzyme implicated in various nuclear processes including elongation and pre-mRNA 3'-end formation. Toward this end, we characterized monoclonal antibodies commonly employed to study CTD phosphorylation patterns and found that the H5 monoclonal antibody reacts with CTD species phosphorylated at Ser2 and/or Ser5. We therefore used antibody-independent methods to study CTDK-I, and we found that CTDK-I phosphorylates Ser5 of the CTD if the CTD substrate is either unphosphorylated or prephosphorylated at Ser2. When Ser5 is already phosphorylated, CTDK-I phosphorylates Ser2 of the CTD. We also observed that CTDK-I efficiently generates doubly phosphorylated CTD repeats; CTD substrates that already contain Ser2-PO(4) or Ser5-PO(4) are more readily phosphorylated CTDK-I than unphosphorylby ated CTD substrates.

Authors
Jones, JC; Phatnani, HP; Haystead, TA; MacDonald, JA; Alam, SM; Greenleaf, AL
MLA Citation
Jones, JC, Phatnani, HP, Haystead, TA, MacDonald, JA, Alam, SM, and Greenleaf, AL. "C-terminal repeat domain kinase I phosphorylates Ser2 and Ser5 of RNA polymerase II C-terminal domain repeats." J Biol Chem 279.24 (June 11, 2004): 24957-24964.
PMID
15047695
Source
pubmed
Published In
The Journal of biological chemistry
Volume
279
Issue
24
Publish Date
2004
Start Page
24957
End Page
24964
DOI
10.1074/jbc.M402218200

Identifying phosphoCTD-associating proteins.

The C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II is hyperphosphorylated during transcription elongation. The phosphoCTD is known to bind to a subset of RNA processing factors and to several other nuclear proteins, thereby positioning them to efficiently carry out their elongation-linked functions. The authors propose that additional phosphoCTD-associating proteins (PCAPs) exist and describe a systematic biochemical approach for identifying such proteins. A binding probe is generated by using yeast CTD kinase I to exhaustively phosphorylate a CTD fusion protein. This phosphoCTD is used to probe fractionated yeast or mammalian extracts in a Far Western protein interaction assay. Putative PCAPs are further purified and identified by mass spectrometry.

Authors
Phatnani, HP; Greenleaf, AL
MLA Citation
Phatnani, HP, and Greenleaf, AL. "Identifying phosphoCTD-associating proteins." Methods Mol Biol 257 (2004): 17-28.
PMID
14769993
Source
pubmed
Published In
Methods in molecular biology (Clifton, N.J.)
Volume
257
Publish Date
2004
Start Page
17
End Page
28
DOI
10.1385/1-59259-750-5:017

Getting a grip on the CTD of pol II

The first structure of a pre-mRNA processing factor bound to heptad repeats from the C-terminal domain of RNA polymerase II is revealed in a crystal of capping guanylyltransferase complexed with a four-repeat phosphopeptide.

Authors
Greenleaf, A
MLA Citation
Greenleaf, A. "Getting a grip on the CTD of pol II." Structure 11.8 (2003): 900-902.
PMID
12906819
Source
scival
Published In
Structure
Volume
11
Issue
8
Publish Date
2003
Start Page
900
End Page
902
DOI
10.1016/S0969-2126(03)00164-3

The RNA polymerase II CTD kinase CTDK-I affects pre-mRNA 3' cleavage/polyadenylation through the processing component Pti1p.

There are several kinases in Saccharomyces cerevisiae that phosphorylate the CTD of RNA polymerase II, but specific and distinct functions of the phospho-CTDs generated by the different kinases are not well understood. A genetic screen for suppressors of loss of yeast CTD kinase I (CTDK-I) function (by deletion of the catalytic subunit gene CTK1) identified PTI1, a potential 3' cleavage/polyadenylation factor. Genetic and physical interactions connect Pti1p to components of CF IA and CF II/CPF, and mutations of PTI1 or CTK1 affect 3' cleavage site choice and transcript abundance of particular genes. Therefore, one important function of the CTDK-I-generated phospho-CTD appears to be the coupling of transcription to 3' processing of pre-mRNAs by a Pti1p-containing complex.

Authors
Skaar, DA; Greenleaf, AL
MLA Citation
Skaar, DA, and Greenleaf, AL. "The RNA polymerase II CTD kinase CTDK-I affects pre-mRNA 3' cleavage/polyadenylation through the processing component Pti1p." Mol Cell 10.6 (December 2002): 1429-1439.
PMID
12504017
Source
pubmed
Published In
Molecular Cell
Volume
10
Issue
6
Publish Date
2002
Start Page
1429
End Page
1439

Hyperphosphorylated C-terminal repeat domain-associating proteins in the nuclear proteome link transcription to DNA/chromatin modification and RNA processing.

Using an interaction blot approach to search in the human nuclear proteome, we identified eight novel proteins that bind the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of RNA polymerase II. Unexpectedly, five of the new phosphoCTD-associating proteins (PCAPs) represent either enzymes that act on DNA and chromatin (topoisomerase I, DNA (cytosine-5) methyltransferase 1, poly(ADP-ribose) polymerase-1) or proteins known to bind DNA (heterogeneous nuclear ribonucleoprotein (hnRNP) U/SAF-A, hnRNP D). The other three PCAPs represent factors involved in pre-mRNA metabolism as anticipated (CA150, NSAP1/hnRNP Q, hnRNP R) (note that hnRNP U/SAF-A and hnRNP D are also implicated in pre-mRNA metabolism). Identifying as PCAPs proteins involved in diverse DNA transactions suggests that the range of phosphoCTD functions extends far beyond just transcription and RNA processing. In view of the activities possessed by the DNA-directed PCAPs, it is likely that the phosphoCTD plays important roles in genome integrity, epigenetic regulation, and potentially nuclear structure. We present a model in which the phosphoCTD association of the PCAPs poises them to act either on the nascent transcript or on the DNA/chromatin template. We propose that the phosphoCTD of elongating RNA polymerase II is a major organizer of nuclear functions.

Authors
Carty, SM; Greenleaf, AL
MLA Citation
Carty, SM, and Greenleaf, AL. "Hyperphosphorylated C-terminal repeat domain-associating proteins in the nuclear proteome link transcription to DNA/chromatin modification and RNA processing." Mol Cell Proteomics 1.8 (August 2002): 598-610.
PMID
12376575
Source
pubmed
Published In
Molecular & cellular proteomics : MCP
Volume
1
Issue
8
Publish Date
2002
Start Page
598
End Page
610

Co-transcriptional splicing of pre-messenger RNAs: considerations for the mechanism of alternative splicing.

Nascent transcripts are the true substrates for many splicing events in mammalian cells. In this review we discuss transcription, splicing, and alternative splicing in the context of co-transcriptional processing of pre-mRNA. The realization that splicing occurs co-transcriptionally requires two important considerations: First, the cis-acting elements in the splicing substrate are synthesized at different times in a 5' to 3' direction. This dynamic view of the substrate implies that in a 100 kb intron the 5' splice site will be synthesized as much as an hour before the 3' splice site. Second, the transcription machinery and the splicing machinery, which are both complex and very large, are working in close proximity to each other. It is therefore likely that these two macromolecular machines interact, and recent data supporting this notion is discussed. We propose a model for co-transcriptional pre-mRNA processing that incorporates the concepts of splice site-tethering and dynamic exon definition. Also, we present a dynamic view of the alternative splicing of FGF-R2 and suggest that this view could be generally applicable to many regulated splicing events.

Authors
Goldstrohm, AC; Greenleaf, AL; Garcia-Blanco, MA
MLA Citation
Goldstrohm, AC, Greenleaf, AL, and Garcia-Blanco, MA. "Co-transcriptional splicing of pre-messenger RNAs: considerations for the mechanism of alternative splicing." Gene 277.1-2 (October 17, 2001): 31-47. (Review)
PMID
11602343
Source
pubmed
Published In
Gene
Volume
277
Issue
1-2
Publish Date
2001
Start Page
31
End Page
47

Phosphorylation of RNA polymerase II CTD fragments results in tight binding to the WW domain from the yeast prolyl isomerase Ess1.

The yeast prolyl isomerase, Ess1, has recently been shown to interact via its WW domain with the hyperphosphorylated form of the RNA polymerase II C-terminal domain (CTD). We have investigated folding of the Ess1 WW domain and its binding to peptides representing the CTD by circular dichroism and fluorescence. Ess1 WW folds and unfolds reversibly, but in the absence of ligand is only marginally stable with a melting temperature of 19 degrees C. The WW domain is stabilized by the addition of anionic ligands, namely, chloride, inorganic phosphate, phosphoserine, and phosphorylated CTD peptides. Dissociation constants were measured to be 70--100 microM for CTD peptides phosphorylated at one serine, and 16--21 microM for peptides with two or more phosphorylated serines. Weaker or no affinity was observed for nonphosphorylated CTD peptides. There is surprisingly little difference in the affinity for peptides phosphorylated at Ser 2 or Ser 5 of the consensus repeat, or for peptides with different patterns of multiple phosphorylation. The binding of Ess1 to phosphorylated CTD peptides is consistent with a model wherein the WW domain positions Ess1 to catalyze isomerization of the many pSer--Pro peptide bonds in the phosphorylated CTD. We suggest that cis/trans isomerization of prolyl peptide bonds plays a crucial role in CTD function during eukaryotic transcription.

Authors
Myers, JK; Morris, DP; Greenleaf, AL; Oas, TG
MLA Citation
Myers, JK, Morris, DP, Greenleaf, AL, and Oas, TG. "Phosphorylation of RNA polymerase II CTD fragments results in tight binding to the WW domain from the yeast prolyl isomerase Ess1." Biochemistry 40.29 (July 24, 2001): 8479-8486.
PMID
11456485
Source
pubmed
Published In
Biochemistry
Volume
40
Issue
29
Publish Date
2001
Start Page
8479
End Page
8486

Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription.

The C-terminal domain (CTD) of the large subunit of RNA polymerase II plays a role in transcription and RNA processing. Yeast ESS1, a peptidyl-prolyl cis/trans isomerase, is involved in RNA processing and can associate with the CTD. Using several types of assays we could not find any evidence of an effect of Pin1, the human homolog of ESS1, on transcription by RNA polymerase II in vitro or on the expression of a reporter gene in vivo. However, an inhibitor of Pin1, 5-hydroxy-1,4-naphthoquinone (juglone), blocked transcription by RNA polymerase II. Unlike N-ethylmaleimide, which inhibited all phases of transcription by RNA polymerase II, juglone disrupted the formation of functional preinitiation complexes by modifying sulfhydryl groups but did not have any significant effect on either initiation or elongation. Both RNA polymerases I and III, but not T7 RNA polymerase, were inhibited by juglone. The primary target of juglone has not been unambiguously identified, although a site on the polymerase itself is suggested by inhibition of RNA polymerase II during factor-independent transcription of single-stranded DNA. Because of its unique inhibitory properties juglone should prove useful in studying transcription in vitro.

Authors
Chao, SH; Greenleaf, AL; Price, DH
MLA Citation
Chao, SH, Greenleaf, AL, and Price, DH. "Juglone, an inhibitor of the peptidyl-prolyl isomerase Pin1, also directly blocks transcription." Nucleic Acids Res 29.3 (February 1, 2001): 767-773.
PMID
11160900
Source
pubmed
Published In
Nucleic Acids Research
Volume
29
Issue
3
Publish Date
2001
Start Page
767
End Page
773

The splicing factor, Prp40, binds the phosphorylated carboxyl-terminal domain of RNA polymerase II.

We showed previously that the WW domain of the prolyl isomerase, Ess1, can bind the phosphorylated carboxyl-terminal domain (phospho-CTD) of the largest subunit of RNA Polymerase II. Analysis of phospho-CTD binding by four other WW domain-containing Saccharomyces cerevisiae proteins indicates the splicing factor, Prp40, and the RNA polymerase II ubiquitin ligase, Rsp5, can also bind the phospho-CTD. The identification of Prp40 as a phospho-CTD binding protein represents the first demonstration of direct interaction between a documented splicing factor and the phospho-CTD. Domain dissection studies reveal that phospho-CTD binding occurs at multiple locations in Prp40, including sites in both the WW and FF domain regions. Because the conserved repeats of the CTD make it an ideal ligand for multi-site binding events, the implications of multi-site binding are discussed. Our data suggest a mechanism by which the phospho-CTD of elongating RNA polymerase II facilitates commitment complex formation by juxtaposing the 5' and 3' splice sites.

Authors
Morris, DP; Greenleaf, AL
MLA Citation
Morris, DP, and Greenleaf, AL. "The splicing factor, Prp40, binds the phosphorylated carboxyl-terminal domain of RNA polymerase II." J Biol Chem 275.51 (December 22, 2000): 39935-39943.
PMID
10978320
Source
pubmed
Published In
The Journal of biological chemistry
Volume
275
Issue
51
Publish Date
2000
Start Page
39935
End Page
39943
DOI
10.1074/jbc.M004118200

Protein-interaction modules that organize nuclear function: FF domains of CA150 bind the phosphoCTD of RNA polymerase II.

An approach for purifying nuclear proteins that bind directly to the hyperphosphorylated C-terminal repeat domain (CTD) of RNA polymerase II was developed and used to identify one human phosphoCTD-associating protein as CA150. CA150 is a nuclear factor implicated in transcription elongation. Because the hyperphosphorylated CTD is a feature of actively transcribing RNA polymerase II (Pol II), phosphoCTD (PCTD) binding places CA150 in a location appropriate for performing a role in transcription elongation-related events. Several recombinant segments of CA150 bound the PCTD. Predominant binding is mediated by the portion of CA150 containing six FF domains, compact modules of previously unknown function. In fact, small recombinant proteins containing the fifth FF domain bound the PCTD. PCTD binding is the first specific function assigned to an FF domain. As FF domains are found in a variety of nuclear proteins, it is likely that some of these proteins are also PCTD-associating proteins. Thus FF domains appear to be compact protein-interaction modules that, like WW domains, can be evolutionarily shuffled to organize nuclear function.

Authors
Carty, SM; Goldstrohm, AC; Suñé, C; Garcia-Blanco, MA; Greenleaf, AL
MLA Citation
Carty, SM, Goldstrohm, AC, Suñé, C, Garcia-Blanco, MA, and Greenleaf, AL. "Protein-interaction modules that organize nuclear function: FF domains of CA150 bind the phosphoCTD of RNA polymerase II." Proc Natl Acad Sci U S A 97.16 (August 1, 2000): 9015-9020.
PMID
10908677
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
97
Issue
16
Publish Date
2000
Start Page
9015
End Page
9020
DOI
10.1073/pnas.160266597

Novel interactions with the phospho-CTD of RNA polymerase II: Connecting transcription with DNA remodeling.

Authors
Carty, SM; Wilborn, M; Greenleaf, AL
MLA Citation
Carty, SM, Wilborn, M, and Greenleaf, AL. "Novel interactions with the phospho-CTD of RNA polymerase II: Connecting transcription with DNA remodeling." FASEB JOURNAL 14.8 (May 11, 2000): A1582-A1582.
Source
wos-lite
Published In
The FASEB journal : official publication of the Federation of American Societies for Experimental Biology
Volume
14
Issue
8
Publish Date
2000
Start Page
A1582
End Page
A1582

Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II.

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

Authors
Rodriguez, CR; Cho, EJ; Keogh, MC; Moore, CL; Greenleaf, AL; Buratowski, S
MLA Citation
Rodriguez, CR, Cho, EJ, Keogh, MC, Moore, CL, Greenleaf, AL, and Buratowski, S. "Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II." Mol Cell Biol 20.1 (January 2000): 104-112.
PMID
10594013
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
20
Issue
1
Publish Date
2000
Start Page
104
End Page
112

Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation.

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.

Authors
Morris, DP; Phatnani, HP; Greenleaf, AL
MLA Citation
Morris, DP, Phatnani, HP, and Greenleaf, AL. "Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation." J Biol Chem 274.44 (October 29, 1999): 31583-31587.
PMID
10531363
Source
pubmed
Published In
The Journal of biological chemistry
Volume
274
Issue
44
Publish Date
1999
Start Page
31583
End Page
31587

Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II.

The carboxyl-terminal domain (CTD) of the largest RNA polymerase II (pol II) subunit is a target for extensive phosphorylation in vivo. Using in vitro kinase assays it was found that several different protein kinases can phosphorylate the CTD including the transcription factor IIH-associated CDK-activating CDK7 kinase (R. Roy, J. P. Adamczewski, T. Seroz, W. Vermeulen, J. P. Tassan, L. Schaeffer, E. A. Nigg, J. H. Hoeijmakers, and J. M. Egly, 1994, Cell 79, 1093-1101). Here we report the colocalization of CDK7 and the phosphorylated form of CTD (phosphoCTD) to actively transcribing genes in intact salivary gland cells of Chironomus tentans. Following a heat-shock treatment, both CDK7 and pol II staining disappear from non-heat-shock genes concomitantly with the abolishment of transcriptional activity of these genes. In contrast, the actively transcribing heat-shock genes, manifested as chromosomal puff 5C on chromosome IV (IV-5C), stain intensely for phosphoCTD, but are devoid of CDK7. Furthermore, the staining of puff IV-5C with anti-PCTD antibodies was not detectably influenced by the TFIIH kinase and transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Following heat-shock treatment, the transcription of non-heat-shock genes was completely eliminated, while newly formed heat-shock gene transcripts emerged in a DRB-resistant manner. Thus, heat shock in these cells induces a rapid clearance of CDK7 from the non-heat-shock genes, indicating a lack of involvement of CDK7 in the induction and function of the heat-induced genes. The results taken together suggest the existence of heat-shock-specific CTD phosphorylation in living cells. This phosphorylation is resistant to DRB treatment, suggesting that not only phosphorylation but also transcription of heat-shock genes is DRB resistant and that CDK7 in heat shock cells is not associated with TFIIH.

Authors
Egyházi, E; Ossoinak, A; Lee, JM; Greenleaf, AL; Mäkelä, TP; Pigon, A
MLA Citation
Egyházi, E, Ossoinak, A, Lee, JM, Greenleaf, AL, Mäkelä, TP, and Pigon, A. "Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II." Exp Cell Res 242.1 (July 10, 1998): 211-221.
PMID
9665818
Source
pubmed
Published In
Experimental Cell Research
Volume
242
Issue
1
Publish Date
1998
Start Page
211
End Page
221
DOI
10.1006/excr.1998.4112

Protonation of the neutral repeats of the RNA polymerase II CTD.

The CTD (carboxy-terminal repeat domain) of the largest subunit of RNA Polymerase II in most eukaryotes consists of from 26 to 52 seven amino acid repeats, the consensus sequence of which is YSPTSPS. Even though this consensus repeat does not contain residues that are normally protonated under the conditions used for positive ion electrospray mass spectrometry, we find that the CTD acquires about one proton per repeat when analyzed by this procedure. We have termed this phenomenon superprotonation. Superprotonation is apparently a property of the consensus sequence as the repeat peptide, (YSPTSPS)4, is superprotonated whereas other proteins and the repeat peptides (YSPTSPK)4, (YSPTSPR)4 and (YSPTAPR)4 are not. The highly conserved nature of the contiguous consensus repeats in organisms ranging from yeast to mammals implies that the functionally significant behavior of the domain is easily perturbed. We propose that CTD superprotonation is a manifestation of a unique biophysical property that will influence and could be the basis for consensus repeat function in vivo.

Authors
Morris, DP; Stevens, RD; Greenleaf, AL
MLA Citation
Morris, DP, Stevens, RD, and Greenleaf, AL. "Protonation of the neutral repeats of the RNA polymerase II CTD." Biochem Biophys Res Commun 245.1 (April 7, 1998): 53-58.
PMID
9535782
Source
pubmed
Published In
Biochemical and Biophysical Research Communications
Volume
245
Issue
1
Publish Date
1998
Start Page
53
End Page
58
DOI
10.1006/bbrc.1998.8373

Assaying CTD kinases in vitro and phosphorylation-modulated properties of RNA polymerase II in vivo.

The functional properties of RNA polymerase II are modulated by hyperphosphorylation of its unique C-terminal repeat domain (CTD). A number of enzymes with CTD kinase activity have been identified, and correlations between CTD phosphorylation and RNA polymerase II function have been made. Here we describe methods for assaying CTD kinases and for characterizing them enzymologically. In addition we present approaches for studying phosphorylation-mediated behavior of chromosome-associated RNA polymerase II by using CTD-directed, phosphorylation state-sensitive antibodies and in situ localization techniques. The methods described here should, in conjunction with genetic approaches, contribute to elucidating the physiological roles of CTD kinases.

Authors
Morris, DP; Lee, JM; Sterner, DE; Brickey, WJ; Greenleaf, AL
MLA Citation
Morris, DP, Lee, JM, Sterner, DE, Brickey, WJ, and Greenleaf, AL. "Assaying CTD kinases in vitro and phosphorylation-modulated properties of RNA polymerase II in vivo." Methods 12.3 (July 1997): 264-275. (Review)
PMID
9237170
Source
pubmed
Published In
Methods
Volume
12
Issue
3
Publish Date
1997
Start Page
264
End Page
275
DOI
10.1006/meth.1997.0478

Modulation of RNA polymerase II elongation efficiency by C-terminal heptapeptide repeat domain kinase I.

Hyperphosphorylation of the C-terminal heptapeptide repeat domain (CTD) of the RNA polymerase II largest subunit has been suggested to play a key role in regulating transcription initiation and elongation. To facilitate investigating functional consequences of CTD phosphorylation we developed new templates, the double G-less cassettes, which make it possible to assay simultaneously the level of initiation and the efficiency of elongation. Using these templates, we examined the effects of yeast CTD kinase I or CTD kinase inhibitors on transcription and CTD phosphorylation in HeLa nuclear extracts. Our results showed that polymerase II elongation efficiency and CTD phosphorylation are greatly reduced by CTD kinase inhibitors, whereas both are greatly increased by CTD kinase I; in contrast, transcription initiation is much less affected. These results demonstrate that CTD kinase I modulates the elongation efficiency of RNA polymerase II and are consistent with the idea that one function of CTD phosphorylation is to promote effective production of long transcripts by stimulating the elongation efficiency of RNA polymerase II.

Authors
Lee, JM; Greenleaf, AL
MLA Citation
Lee, JM, and Greenleaf, AL. "Modulation of RNA polymerase II elongation efficiency by C-terminal heptapeptide repeat domain kinase I." J Biol Chem 272.17 (April 25, 1997): 10990-10993.
PMID
9110987
Source
pubmed
Published In
The Journal of biological chemistry
Volume
272
Issue
17
Publish Date
1997
Start Page
10990
End Page
10993

Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract.

Analyses of Drosophila cells have revealed that RNA polymerase II is paused in a region 20 to 40 nucleotides downstream from the transcription start site of the hsp70 heat shock gene when the gene is not transcriptionally active. We have developed a cell-free system that reconstitutes this promoter-proximal pausing. The paused polymerase has been detected by monitoring the hyperreactivity of thymines in the transcription bubble toward potassium permanganate. The pattern of permanganate reactivity for the hsp70 promoter in the reconstituted system matches the pattern found on the promoter after it has been introduced back into files by P-element-mediated transposition. Matching patterns of permanganate reactivity are also observed for a non-heat shock promoter, the histone H3 promoter. Further analysis of the hsp70 promoter in the reconstituted system reveals that pausing does not depend on sequence-specific interactions located immediately downstream from the pause site. Sequences upstream from the TATA box influence the recruitment of polymerase rather than the efficiency of pausing. Kinetic analysis indicates that the polymerase rapidly enters the paused state and remains stably in this state for at least 25 min. Further analysis shows that the paused polymerase will initially resume elongation when Sarkosyl is added but loses this capacity within minutes of pausing. Using an alpha-amanitin-resistant polymerase, we provide evidence that promoter-proximal pausing does not require the carboxy-terminal domain of the polymerase.

Authors
Li, B; Weber, JA; Chen, Y; Greenleaf, AL; Gilmour, DS
MLA Citation
Li, B, Weber, JA, Chen, Y, Greenleaf, AL, and Gilmour, DS. "Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract." Mol Cell Biol 16.10 (October 1996): 5433-5443.
PMID
8816456
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
16
Issue
10
Publish Date
1996
Start Page
5433
End Page
5443

Drosophila RNA polymerase II mutants that affect transcription elongation.

We have examined the properties of two Drosophila RNA polymerase II mutants, C4 and S1, during elongation, pyrophosphorolysis, and DmS-II-stimulated transcript cleavage. The C4 and S1 mutants contain a single amino acid substitution in the largest and second largest subunits, respectively. Compared with wild type, C4 had a lower elongation rate and was less efficient at reading through intrinsic elongation blocks. S1 had a higher elongation rate than wild type and was more efficient at reading through the same blocks. During elongation, C4 and wild type responded similarly to DmS-II and NH4+ whereas the S1 mutant was less responsive to both. Differences between the two mutants also appeared during DmS-II-mediated transcript cleavage and pyrophosphorolysis. During extended pyrophosphorolysis, S1 polymerase was fastest and C4 polymerase was slowest at generating the final pattern of shortened transcripts. S1 and wild type were equal in the rate of extended DmS-II mediated transcript cleavage, and C4 was slower. Our results suggest that the S1 mutation increases the time spent by the polymerase in elongation competent mode and that the C4 mutation may affect the movement of the polymerase.

Authors
Chen, Y; Chafin, D; Price, DH; Greenleaf, AL
MLA Citation
Chen, Y, Chafin, D, Price, DH, and Greenleaf, AL. "Drosophila RNA polymerase II mutants that affect transcription elongation." J Biol Chem 271.11 (March 15, 1996): 5993-5999.
PMID
8626382
Source
pubmed
Published In
The Journal of biological chemistry
Volume
271
Issue
11
Publish Date
1996
Start Page
5993
End Page
5999

Phosphorylation dependence of the initiation of productive transcription of Balbiani ring 2 genes in living cells.

Using polytene chromosomes of salivary gland cells of Chironomus tentans, phosphorylation state-sensitive antibodies and the transcription and protein kinase inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), we have visualized the chromosomal distribution of RNA polymerase II (pol II) with hypophosphorylated (pol IIA) and hyperphosphorylated (pol II0) carboxyl-terminal repeat domain (CTD). DRB blocks labeling of the CTD with 32Pi within minutes of its addition, and nuclear pol II0 is gradually converted to IIA; this conversion parallels the reduction in transcription of protein-coding genes. DRB also alters the chromosomal distribution of II0: there is a time-dependent clearance from chromosomes of phosphoCTD (PCTD) after addition of DRB, which coincides in time with the completion and release of preinitiated transcripts. Furthermore, the staining of smaller transcription units is abolished before that of larger ones. The staining pattern of chromosomes with anti-CTD antibodies is not detectably influenced by the DRB treatment, indicating that hypophosphorylated pol IIA is unaffected by the transcription inhibitor. Microinjection of synthetic heptapeptide repeats, anti-CTD and anti-PCTD antibodies into salivary gland nuclei hampered the transcription of BR2 genes, indicating the requirement for CTD and PCTD in transcription in living cells. The results demonstrate that in vivo the protein kinase effector DRB shows parallel effects on an early step in gene transcription and the process of pol II hyperphosphorylation. Our observations are consistent with the proposal that the initiation of productive RNA synthesis is CTD-phosphorylation dependent and also with the idea that the gradual dephosphorylation of transcribing pol II0 is coupled to the completion of nascent pol II gene transcripts.

Authors
Egyházi, E; Ossoinak, A; Pigon, A; Holmgren, C; Lee, JM; Greenleaf, AL
MLA Citation
Egyházi, E, Ossoinak, A, Pigon, A, Holmgren, C, Lee, JM, and Greenleaf, AL. "Phosphorylation dependence of the initiation of productive transcription of Balbiani ring 2 genes in living cells." Chromosoma 104.6 (March 1996): 422-433.
PMID
8601337
Source
pubmed
Published In
Chromosoma
Volume
104
Issue
6
Publish Date
1996
Start Page
422
End Page
433

DIFFERENTIAL INHIBITORY EFFECT OF DRB ON THE TRANSCRIPTIONAL ACTIVITY AND PHOSPHORYLATION OF RNA-POLYMERASE-II IN HEAT-SHOCKED AND NONSHOCKED CELLS

Authors
EGYHAZI, E; OSSOINAK, A; PIGON, A; LEE, JM; GREENLEAF, AL
MLA Citation
EGYHAZI, E, OSSOINAK, A, PIGON, A, LEE, JM, and GREENLEAF, AL. "DIFFERENTIAL INHIBITORY EFFECT OF DRB ON THE TRANSCRIPTIONAL ACTIVITY AND PHOSPHORYLATION OF RNA-POLYMERASE-II IN HEAT-SHOCKED AND NONSHOCKED CELLS." MOLECULAR BIOLOGY OF THE CELL 6 (November 1995): 439-439.
Source
wos-lite
Published In
Molecular Biology of the Cell
Volume
6
Publish Date
1995
Start Page
439
End Page
439

The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex.

Saccharomyces cerevisiae CTDK-I is a protein kinase complex that specifically and efficiently hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II and is composed of three subunits of 58, 38, and 32 kDa. The kinase is essential in vivo for normal phosphorylation of the CTD and for normal growth and differentiation. We have now cloned the genes for the two smaller kinase subunits, CTK2 and CTK3, and found that they form a unique, divergent cyclin-cyclin-dependent kinase complex with the previously characterized largest subunit protein CTK1, a cyclin-dependent kinase homolog. The CTK2 gene encodes a cyclin-related protein with limited homology to cyclin C, while CTK3 shows no similarity to other known proteins. Copurification of the three gene products with each other and CTDK-I activity by means of conventional chromatography and antibody affinity columns has verified their participation in the complex in vitro. In addition, null mutations of each of the genes and all combinations thereof conferred very similar growth-impaired, cold-sensitive phenotypes, consistent with their involvement in the same function in vivo. These characterizations and the availability of all of the genes encoding CTDK-I and reagents derivable from them will facilitate investigations into CTD phosphorylation and its functional consequences both in vivo and in vitro.

Authors
Sterner, DE; Lee, JM; Hardin, SE; Greenleaf, AL
MLA Citation
Sterner, DE, Lee, JM, Hardin, SE, and Greenleaf, AL. "The yeast carboxyl-terminal repeat domain kinase CTDK-I is a divergent cyclin-cyclin-dependent kinase complex." Mol Cell Biol 15.10 (October 1995): 5716-5724.
PMID
7565723
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
15
Issue
10
Publish Date
1995
Start Page
5716
End Page
5724

Functional studies of the carboxy-terminal repeat domain of Drosophila RNA polymerase II in vivo.

To understand the in vivo function of the unique and conserved carboxy-terminal repeat domain (CTD) of RNA polymerase II largest subunit (RpII215), we have studied RNA polymerase II biosynthesis, activity and genetic function in Drosophila RpII215 mutants that possessed all (C4), half (W81) or none (IIt) of the CTD repeats. We have discovered that steady-state mRNA levels from transgenes encoding a fully truncated, CTD-less subunit (IIt) are essentially equal to wild-type levels, whereas the levels of the CTD-less subunit itself and the amount of polymerase harboring it (Pol IIT) are significantly lower than wild type. In contrast, for the half-CTD mutant (W81), steady-state mRNA levels are somewhat lower than for wild type or IIt, while W81 subunit and polymerase amounts are much less than wild type. Finally, we have tested genetically the ability of CTD mutants to complement (rescue) partially functional RpII215 alleles and have found that IIt fails to complement whereas W81 complements partially to completely. These results suggest that removal of the entire CTD renders polymerase completely defective in vivo, whereas eliminating half of the CTD results in a polymerase with significant in vivo activity.

Authors
Brickey, WJ; Greenleaf, AL
MLA Citation
Brickey, WJ, and Greenleaf, AL. "Functional studies of the carboxy-terminal repeat domain of Drosophila RNA polymerase II in vivo." Genetics 140.2 (June 1995): 599-613.
PMID
7498740
Source
pubmed
Published In
Genetics
Volume
140
Issue
2
Publish Date
1995
Start Page
599
End Page
613

Identifying a transcription factor interaction site on RNA polymerase II.

We have generated a series of fusion proteins carrying portions of subunit IIc, the second largest subunit of Drosophila RNA polymerase I, and have used them in a domain interference assay to identify a fragment of the IIc subunit that carries the binding site for a basal transcription factor. Fusion proteins carrying a subunit IIc fragment spanning residues Ala519-Gly992 strongly inhibit promoter-driven transcription in both unfractionated nuclear extracts and in reconstituted systems. The same fusion proteins similarly inhibit dTFIIF stimulation of Pol II elongation on dC-tailed templates, suggesting that the IIc(A519-G992) fragment, which carries conserved regions D-H, interferes with transcription by binding to dTFIIF. Finally, dTFIIF can be specifically cross-linked to a GST-IIc(A519-G992) fusion protein or to subunit IIc in intact Pol II.

Authors
Skantar, AM; Greenleaf, AL
MLA Citation
Skantar, AM, and Greenleaf, AL. "Identifying a transcription factor interaction site on RNA polymerase II." Gene Expr 5.1 (1995): 49-69.
PMID
7488860
Source
pubmed
Published In
Gene expression
Volume
5
Issue
1
Publish Date
1995
Start Page
49
End Page
69

RNA-POLYMERASE-II CTD PHOSPHORYLATION IN-VIVO

Authors
WEEKS, JR; HARDIN, S; SHEN, JJ; LEE, JM; GREENLEAF, AL
MLA Citation
WEEKS, JR, HARDIN, S, SHEN, JJ, LEE, JM, and GREENLEAF, AL. "RNA-POLYMERASE-II CTD PHOSPHORYLATION IN-VIVO." JOURNAL OF CELLULAR BIOCHEMISTRY (February 13, 1994): 3-3.
Source
wos-lite
Published In
Journal of Cellular Biochemistry
Publish Date
1994
Start Page
3
End Page
3

Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II is essential in vivo, and is found in either an unphosphorylated (IIa) or hyperphosphorylated (IIo) form. The Drosophila uninduced hsp70 and hsp26 genes, and the constitutively expressed β-1 tubulin and Gapdh-2 genes, contain an RNA polymerase II complex which pauses after synthesizing a short transcript. We report here that, using an in vivo ultraviolet crosslinking technique and antibodies directed against the IIa and IIo forms of the CTD, these paused polymerases have an unphosphorylated CTD. For genes containing a 5' paused polymerase, passage of the paused RNA polymerase into an elongationally competent mode in vivo coincides with phosphorylation of the CTD. Also, the level of phosphorylation of the CTD of elongating polymerases is shown not to be related to the level of transcription, but is promoter specific.

Authors
O'Brien, T; Hardin, S; Greenleaf, A; Lis, JT
MLA Citation
O'Brien, T, Hardin, S, Greenleaf, A, and Lis, JT. "Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation." Nature 370.6484 (1994): 75-77.
PMID
8015613
Source
scival
Published In
Nature
Volume
370
Issue
6484
Publish Date
1994
Start Page
75
End Page
77
DOI
10.1038/370075a0

A positive addition to a negative tail's tale.

Authors
Greenleaf, AL
MLA Citation
Greenleaf, AL. "A positive addition to a negative tail's tale." Proc Natl Acad Sci U S A 90.23 (December 1, 1993): 10896-10897. (Review)
PMID
7504285
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
90
Issue
23
Publish Date
1993
Start Page
10896
End Page
10897

Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing.

To investigate functional differences between RNA polymerases IIA and IIO (Pol IIA and Pol IIO), with hypo- and hyperphosphorylated carboxy-terminal repeat domains (CTDs), respectively, we have visualized the in vivo distributions of the differentially phosphorylated forms of Pol II on Drosophila polytene chromosomes. Using phosphorylation state-sensitive antibodies and immunofluorescence microscopy with digital imaging, we find Pol IIA and Pol IIO arrayed in markedly different, locus- and condition-specific patterns. Major ecdysone-induced puffs, for example, stain exclusively for Pol IIO, indicating that hyperphosphorylated Pol II is the transcriptionally active form of the enzyme on these genes. In striking contrast, induced heat shock puffs stain strongly for both Pol IIA and Pol IIO, suggesting that heat shock genes are transcribed by a mixture of hypo- and hyperphosphorylated forms of Pol II. At the insertion sites of a transposon carrying a hybrid hsp70-lacZ transgene, we observe only Pol IIA before heat shock induction, consistent with the idea that Pol II arrested on the hsp70 gene is form IIA. After a 90-sec heat shock, we detect heat shock factor (HSF) at the transposon insertion sites; and after a 5-min shock its spatial distribution on the induced transgene puffs is clearly resolved from that of Pol II. Finally, using antibodies to hnRNP proteins and splicing components, we have discerned an apparent overall correlation between the presence and processing of nascent transcripts and the presence of Pol IIO.

Authors
Weeks, JR; Hardin, SE; Shen, J; Lee, JM; Greenleaf, AL
MLA Citation
Weeks, JR, Hardin, SE, Shen, J, Lee, JM, and Greenleaf, AL. "Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing." Genes Dev 7.12A (December 1993): 2329-2344.
PMID
8253380
Source
pubmed
Published In
Genes & development
Volume
7
Issue
12A
Publish Date
1993
Start Page
2329
End Page
2344

Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes.

We have mapped a number of mutations at the DNA sequence level in genes encoding the largest (RpII215) and second-largest (RpII140) subunits of Drosophila melanogaster RNA polymerase II. Using polymerase chain reaction (PCR) amplification and single-strand conformation polymorphism (SSCP) analysis, we detected 12 mutations from 14 mutant alleles (86%) as mobility shifts in nondenaturing gel electrophoresis, thus localizing the mutations to the corresponding PCR fragments of about 350 bp. We then determined the mutations at the DNA sequence level by directly subcloning the PCR fragments and sequencing them. The five mapped RpII140 mutations clustered in a C-terminal portion of the second-largest subunit, indicating the functional importance of this region of the subunit. The RpII215 mutations were distributed more broadly, although six of eight clustered in a central region of the subunit. One notable mutation that we localized to this region was the alpha-amanitin-resistant mutation RpII215C4, which also affects RNA chain elongation in vitro. RpII215C4 mapped to a position near the sites of corresponding mutations in mouse and in Caenorhabditis elegans genes, reinforcing the idea that this region is involved in amatoxin binding and transcript elongation. We also mapped mutations in both RpII215 and RpII140 that cause a developmental defect known as the Ubx effect. The clustering of these mutations in each gene suggests that they define functional domains in each subunit whose alteration induces the mutant phenotype.

Authors
Chen, Y; Weeks, J; Mortin, MA; Greenleaf, AL
MLA Citation
Chen, Y, Weeks, J, Mortin, MA, and Greenleaf, AL. "Mapping mutations in genes encoding the two large subunits of Drosophila RNA polymerase II defines domains essential for basic transcription functions and for proper expression of developmental genes." Mol Cell Biol 13.7 (July 1993): 4214-4222.
PMID
8321225
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
13
Issue
7
Publish Date
1993
Start Page
4214
End Page
4222

Reverse genetics of Drosophila RNA polymerase II: identification and characterization of RpII140, the genomic locus for the second-largest subunit.

We have used a reverse genetics approach to isolate genes encoding two subunits of Drosophila melanogaster RNA polymerase II. RpII18 encodes the 18-kDa subunit and maps cytogenetically to polytene band region 83A. RpII140 encodes the 140-kDa subunit and maps to polytene band region 88A10:B1,2. Focusing on RpII140, we used in situ hybridization to map this gene to a small subinterval defined by the endpoints of a series of deficiencies impinging on the 88A/B region and showed that it does not represent a previously known genetic locus. Two recently defined complementation groups, A5 and Z6, reside in the same subinterval and thus were candidates for the RpII140 locus. Phenotypes of A5 mutants suggested that they affect RNA polymerase II, in that the lethal phase and the interaction with developmental loci such as Ubx resemble those of mutants in the gene for the largest subunit, RpII215. Indeed, we have achieved complete genetic rescue of representative recessive lethal mutations of A5 with a P-element construct containing a 9.1-kb genomic DNA fragment carrying RpII140. Interestingly, the initial construct also rescued lethal alleles in the neighboring complementation group, Z6, revealing that the 9.1-kb insert carries two genes. Deleting coding region sequences of RpII140, however, yielded a transformation vector that failed to rescue A5 alleles but continued to rescue Z6 alleles. These results strongly support the conclusion that the A5 complementation group is equivalent to the genomic RpII140 locus.

Authors
Hamilton, BJ; Mortin, MA; Greenleaf, AL
MLA Citation
Hamilton, BJ, Mortin, MA, and Greenleaf, AL. "Reverse genetics of Drosophila RNA polymerase II: identification and characterization of RpII140, the genomic locus for the second-largest subunit." Genetics 134.2 (June 1993): 517-529.
PMID
8325487
Source
pubmed
Published In
Genetics
Volume
134
Issue
2
Publish Date
1993
Start Page
517
End Page
529

Positive patches and negative noodles: linking RNA processing to transcription?

A speculative model is presented that proposes specific mechanisms for effecting co-transcriptional splice site selection in pre-mRNAs. The model envisions that certain splicing factors containing arginine-rich, positively charged regions bind via these positive patches to the hyperphosphorylated, negatively charged tail of elongating RNA polymerase II. Thus tethered to the transcription machinery, these splicing factors gain access to signals in nascent transcripts as they emerge from the polymerase.

Authors
Greenleaf, AL
MLA Citation
Greenleaf, AL. "Positive patches and negative noodles: linking RNA processing to transcription?." Trends Biochem Sci 18.4 (April 1993): 117-119.
PMID
8493720
Source
pubmed
Published In
Trends in Biochemical Sciences
Volume
18
Issue
4
Publish Date
1993
Start Page
117
End Page
119

Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: Correlations with gene activity and transcript processing

To investigate functional differences between RNA polymerases IIA and II0 (Pol HA and Pol II0), with hypo- and hyperphosphorylated carboxy-terminal repeat domains (CTDs), respectively, we have visualized the in vivo distributions of the differentially phosphorylated forms of Pol II on Drosophila polytene chromosomes. Using phosphorylation state-sensitive antibodies and immunofluorescence microscopy with digital imaging, we find Pol IIA and Pol II0 arrayed in markedly different, locus- and condition- specific patterns. Major ecdysone-induced puffs, for example, stain exclusively for Pol II0, indicating that hyperphosphorylated Pol II is the transcriptionally active form of the enzyme on these genes. In striking contrast, induced heat shock puffs stain strongly for both Pol IIA and Pol II0, suggesting that heat shock genes are transcribed by a mixture of hypo- and hyperphosphorylated forms of Pol II. At the insertion sites of a transposon carrying a hybrid hsp70-lacZ transgene, we observe only Pol IIA before heat shock induction, consistent with the idea that Pol II arrested on the hsp70 gene is form IIA. After a 90-sec heat shock, we detect heat shock factor (HSF) at the transposon insertion sites; and after a 5-min shock its spatial distribution on the induced transgene puffs is clearly resolved from that of Pol II. Finally, using antibodies to hnRNP proteins and splicing components, we have discerned an apparent overall correlation between the presence and processing of nascent transcripts and the presence of Pol II0.

Authors
Weeks, JR; Hardin, SE; Shen, J; Lee, JM; Greenleaf, AL
MLA Citation
Weeks, JR, Hardin, SE, Shen, J, Lee, JM, and Greenleaf, AL. "Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: Correlations with gene activity and transcript processing." Genes and Development 7.12 A (1993): 2329-2344.
Source
scival
Published In
Genes and Development
Volume
7
Issue
12 A
Publish Date
1993
Start Page
2329
End Page
2344

CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae.

We previously purified a yeast protein kinase that specifically hyperphosphorylates the carboxyl-terminal repeat domain (CTD) of RNA polymerase II largest subunit and showed that this CTD kinase consists of three subunits of 58, 38, and 32 kDa. We have now cloned, sequenced, and characterized CTK1, the gene encoding the 58 kDa alpha subunit. The CTK1 gene product contains a central domain homologous to catalytic subunits of other protein kinases, notably yeast CDC28, suggesting that the 58 kDa subunit is catalytic. Cells that carry a disrupted version of the CTK1 gene lack the characterized CTD kinase activity, grow slowly and are cold-sensitive, demonstrating that the CTK1 gene product is essential for CTD kinase activity and normal growth. While ctk1 mutant cells do contain phosphorylated forms of the RNA polymerase II largest subunit, these forms differ from those found in wild type cells, implicating CTK1 as a component of the physiologically significant CTD phosphorylating machinery. As befitting an enzyme with a nuclear function, the N-terminal region of the CTK1 protein contains a nuclear targeting signal.

Authors
Lee, JM; Greenleaf, AL
MLA Citation
Lee, JM, and Greenleaf, AL. "CTD kinase large subunit is encoded by CTK1, a gene required for normal growth of Saccharomyces cerevisiae." Gene Expr 1.2 (May 1991): 149-167.
PMID
1820212
Source
pubmed
Published In
Gene expression
Volume
1
Issue
2
Publish Date
1991
Start Page
149
End Page
167

The carboxyl-terminal repeat domain of RNA polymerase II is not required for transcription factor Sp1 to function in vitro.

We show that the mammalian transcription Sp1 stimulates accurate transcription in a partially fractionated RNA polymerase II-dependent system from Drosophila cultured cells. Moreover, the extent of stimulation is equal for intact RNA polymerase II (polymerase IIA) and polymerase lacking the unique carboxyl-terminal domain of the largest subunit (polymerase IIB). We conclude that in this system Sp1 interacts with a component of the transcription machinery, other than the carboxyl-terminal domain, which is preserved between mammals and insects.

Authors
Zehring, WA; Greenleaf, AL
MLA Citation
Zehring, WA, and Greenleaf, AL. "The carboxyl-terminal repeat domain of RNA polymerase II is not required for transcription factor Sp1 to function in vitro." J Biol Chem 265.15 (May 25, 1990): 8351-8353.
PMID
2187861
Source
pubmed
Published In
The Journal of biological chemistry
Volume
265
Issue
15
Publish Date
1990
Start Page
8351
End Page
8353

Properties of a Drosophila RNA polymerase II elongation factor.

We have purified from nuclear extracts of Drosophila Kc cells a 36-kDa protein, DmS-II, which has an effect on the elongation properties of RNA polymerase II. DmS-II stimulates RNA polymerase II during the transcription of double-stranded DNA templates when the nonphysiological divalent cation manganese is present. In the presence of physiological mono- and divalent cations, the factor reduces the tendency of RNA polymerase II to pause at specific sites along a dC-tailed template including the major pause encountered after 14 nucleotides have been incorporated. Based on its size and chromatographic properties, as well as its ability to stimulate RNA polymerase II activity in the presence of manganese, the protein seems to be analogous to a factor S-II purified from mouse cells (Sekimizu, K., Kobayashi, N., Mizuno, D., and Natori, S. (1976) Biochemistry 15, 5064-5070). We have used a completely defined system and show that the properties of DmS-II are intrinsic to the factor and not mediated through other factors.

Authors
Sluder, AE; Greenleaf, AL; Price, DH
MLA Citation
Sluder, AE, Greenleaf, AL, and Price, DH. "Properties of a Drosophila RNA polymerase II elongation factor." J Biol Chem 264.15 (May 25, 1989): 8963-8969.
PMID
2722810
Source
pubmed
Published In
The Journal of biological chemistry
Volume
264
Issue
15
Publish Date
1989
Start Page
8963
End Page
8969

A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II.

The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts.

Authors
Lee, JM; Greenleaf, AL
MLA Citation
Lee, JM, and Greenleaf, AL. "A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II." Proc Natl Acad Sci U S A 86.10 (May 1989): 3624-3628.
PMID
2657724
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
86
Issue
10
Publish Date
1989
Start Page
3624
End Page
3628

Dynamic interaction between a Drosophila transcription factor and RNA polymerase II.

We have purified factor 5, a Drosophila RNA polymerase II transcription factor. Factor 5 was found to be required for accurate initiation of transcription from specific promoters and also had a dramatic effect on the elongation properties of RNA polymerase II. Kinetic studies suggested that factor 5 stimulates the elongation rate of RNA polymerase II on a dC-tailed, double-stranded template by reducing the time spent at the numerous pause sites encountered by the polymerase. The factor was found to be composed of two polypeptides (34 and 86 kilodaltons). Both subunits bound tightly to pure RNA polymerase II but were not bound to polymerase in elongation complexes. Our results suggest that factor 5 interacts briefly with the paused polymerase molecules and catalyzes a conformational change in them such that they adopt an elongation-competent conformation.

Authors
Price, DH; Sluder, AE; Greenleaf, AL
MLA Citation
Price, DH, Sluder, AE, and Greenleaf, AL. "Dynamic interaction between a Drosophila transcription factor and RNA polymerase II." Mol Cell Biol 9.4 (April 1989): 1465-1475.
PMID
2725511
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
9
Issue
4
Publish Date
1989
Start Page
1465
End Page
1475

Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila.

We have characterized RpII215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster. DNA sequencing and nuclease S1 analyses provided the primary structure of this gene, its 7 kb RNA and 215 kDa protein products. The amino-terminal 80% of the subunit harbors regions with strong homology to the beta' subunit of Escherichia coli RNA polymerase and to the largest subunits of other eukaryotic RNA polymerases. The carboxyl-terminal 20% of the subunit is composed of multiple repeats of a seven amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The homology domains, as well as the unique carboxyl-terminal structure, are considered in the light of current knowledge of RNA polymerase II and the properties of its largest subunit. Additionally, germline transformation demonstrated that a 9.4 kb genomic DNA segment containing the alpha-amanitin-resistant allele, RpII215C4, includes all sequences required to produce amanitin-resistant transformants.

Authors
Jokerst, RS; Weeks, JR; Zehring, WA; Greenleaf, AL
MLA Citation
Jokerst, RS, Weeks, JR, Zehring, WA, and Greenleaf, AL. "Analysis of the gene encoding the largest subunit of RNA polymerase II in Drosophila." Mol Gen Genet 215.2 (January 1989): 266-275.
PMID
2496296
Source
pubmed
Published In
Molecular & general genetics : MGG
Volume
215
Issue
2
Publish Date
1989
Start Page
266
End Page
275

Heat shock-induced appearance of RNA polymerase II in karyoskeletal protein-enriched (nuclear "matrix") fractions correlates with transcriptional shutdown in Drosophila melanogaster.

Heat shock in vivo or brief incubation at moderately elevated temperatures (15 min at 37 degrees C) in vitro is required for the structural stability of proteinaceous karyoskeletal elements purified from Drosophila melanogaster (McConnell, M., Whalen, A. M., Smith, D. E., and Fisher, P. A. (1987) J. Cell Biol. 105, 1087-1098). We have found that the appearance of the two largest subunits of RNA polymerase II in karyoskeletal preparations is promoted by conditions identical to those which promote in vitro stability of karyoskeletal elements overall. Increased levels of polymerase II in karyoskeletal protein-enriched fractions correlate with decreased levels of nucleotide incorporation in nuclear transcription run-on assays. These results are not easily reconciled with the proposal that putative karyoskeletal elements such as the internal nuclear "matrix" are sites of active transcription in vivo.

Authors
Fisher, PA; Lin, L; McConnell, M; Greenleaf, A; Lee, JM; Smith, DE
MLA Citation
Fisher, PA, Lin, L, McConnell, M, Greenleaf, A, Lee, JM, and Smith, DE. "Heat shock-induced appearance of RNA polymerase II in karyoskeletal protein-enriched (nuclear "matrix") fractions correlates with transcriptional shutdown in Drosophila melanogaster." Journal of Biological Chemistry 264.6 (1989): 3464-3469.
PMID
2492538
Source
scival
Published In
Journal of Biological Chemistry
Volume
264
Issue
6
Publish Date
1989
Start Page
3464
End Page
3469

Elongation by Drosophila RNA polymerase II. Transcription of 3'-extended DNA templates.

RNA polymerase II will efficiently initiate transcription on linear duplex DNA which has been extended at its 3' ends by the addition of short stretches of polydeoxycytidine (Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295). We have used such dC-tailed templates to identify factors affecting elongation by Drosophila RNA polymerase II (Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255). While studying these factors we have observed two unexpected characteristics of transcription of the tailed templates. First, we found that RNA polymerase II encountered a strong pause site after the incorporation of 14 nucleotides. This pausing was observed on all templates examined and with RNA polymerase II from a variety of sources. In addition, we found that ammonium ions markedly stimulated the polymerase, increasing both the efficiency with which the enzyme left the 14 base pause site and the subsequent rate of elongation. A factor previously shown to affect transcription of dC-tailed templates (factor 4, Price, D. H., Sluder, A. E., and Greenleaf, A. L. (1987) J. Biol. Chem. 262, 3244-3255) was found to cause transcript displacement and to stimulate the elongation rate approximately 2-fold. This factor copurified with an RNase H activity, and a model is presented for the mechanism of transcript displacement by RNase H. The observations presented here form a basis for further analysis of RNA polymerase II elongation and its modulation by transcription factors. They should also aid in the interpretation of other experiments in which dC-tailed templates are used.

Authors
Sluder, AE; Price, DH; Greenleaf, AL
MLA Citation
Sluder, AE, Price, DH, and Greenleaf, AL. "Elongation by Drosophila RNA polymerase II. Transcription of 3'-extended DNA templates." J Biol Chem 263.20 (July 15, 1988): 9917-9925.
PMID
2454924
Source
pubmed
Published In
The Journal of biological chemistry
Volume
263
Issue
20
Publish Date
1988
Start Page
9917
End Page
9925

The C-terminal repeat domain of RNA polymerase II largest subunit is essential in vivo but is not required for accurate transcription initiation in vitro.

DNA sequence analysis of RpII215, the gene that encodes the Mr215,000 subunit of RNA polymerase II (EC 2.7.7.6) in Drosophila melanogaster, reveals that the 3'-terminal exon includes a region encoding a C-terminal domain composed of 42 repeats of a seven-residue amino acid consensus sequence, Tyr-Ser-Pro-Thr-Ser-Pro-Ser. A hemi- and homozygous lethal P-element insertion into the coding sequence of this domain causes premature translation termination and therefore truncation of the protein, leaving only 20 heptamer repeats. While loss of approximately 50% of the repeat structure in this mutant is a lethal event in vivo, enzyme containing the truncated subunit remains capable of accurate initiation at promoters in vitro. Moreover, treatment of purified intact RNA polymerase II with protease, to remove the entire repeat domain, does not eliminate the enzyme's ability to initiate accurately in vitro. Possible in vivo functions for this unusual protein domain are considered in light of these results.

Authors
Zehring, WA; Lee, JM; Weeks, JR; Jokerst, RS; Greenleaf, AL
MLA Citation
Zehring, WA, Lee, JM, Weeks, JR, Jokerst, RS, and Greenleaf, AL. "The C-terminal repeat domain of RNA polymerase II largest subunit is essential in vivo but is not required for accurate transcription initiation in vitro." Proc Natl Acad Sci U S A 85.11 (June 1988): 3698-3702.
PMID
3131761
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
85
Issue
11
Publish Date
1988
Start Page
3698
End Page
3702

An activity necessary for in vitro transcription is a DNase inhibitor.

A phosphocellulose flowthrough fraction required for accurate transcription in vitro by RNA polymerase II was found to contain a DNase inhibitor which was necessary to maintain template integrity (Price D.H., Sluder A.E. & Greenleaf A.L. (1987) J. Biol. Chem. 262, 3244-3255). Starting with a Drosophila Kc cell nuclear extract, the DNase inhibitory activity has been purified 19,000-fold. In combination with the other necessary fractions, the highly purified inhibitor continues to support reconstruction of transcription. It thus appears to be the only required activity in the original phosphocellulose flowthrough fraction. The inhibitor is a protein which does not bind to DNA or inhibit DNase I, so that it has also been useful in assays for DNA binding proteins in crude, DNase-contaminated fractions.

Authors
Sluder, AE; Price, DH; Greenleaf, AL
MLA Citation
Sluder, AE, Price, DH, and Greenleaf, AL. "An activity necessary for in vitro transcription is a DNase inhibitor." Biochimie 69.11-12 (November 1987): 1199-1205.
PMID
3129025
Source
pubmed
Published In
Biochimie
Volume
69
Issue
11-12
Publish Date
1987
Start Page
1199
End Page
1205

Fractionation of transcription factors for RNA polymerase II from Drosophila Kc cell nuclear extracts.

Drosophila Kc cells were utilized to prepare nuclear extracts in which promoter-containing DNA templates were efficiently transcribed by RNA polymerase II. A combination of fractionation schemes was used to identify and partially purify seven activities (factors) which affected the transcription of four different genes in vitro. Reconstructing specific transcription required exogenous RNA polymerase II in addition to these factors. Moreover, the high efficiency of transcription characteristic of the crude extract was preserved in reconstruction reactions. The methods used are presented in detail. Functions were assigned to several of the factors. One essential factor appeared to affect initiation and displayed chromatographic properties unlike any other Drosophila transcription factor previously described. Two factors specifically affected RNA chain elongation. Another activity was a DNase inhibitor required to preserve template integrity in the fractionated system. The remaining three factors were not absolutely essential but affected the specific in vitro transcription either qualitatively or quantitatively. A comparison of these transcription factors with other Drosophila and mammalian transcription factors is made.

Authors
Price, DH; Sluder, AE; Greenleaf, AL
MLA Citation
Price, DH, Sluder, AE, and Greenleaf, AL. "Fractionation of transcription factors for RNA polymerase II from Drosophila Kc cell nuclear extracts." J Biol Chem 262.7 (March 5, 1987): 3244-3255.
PMID
3818640
Source
pubmed
Published In
The Journal of biological chemistry
Volume
262
Issue
7
Publish Date
1987
Start Page
3244
End Page
3255

Sites of P element insertion and structures of P element deletions in the 5' region of Drosophila melanogaster RpII215.

Several P element insertion and deletion mutations near the 5' end of Drosophila melanogaster RpII215 have been examined by nucleotide sequencing. Two different sites of P element insertion, approximately 90 nucleotides apart, have been detected in this region of the gene. Therefore, including an additional site of P element insertion within the coding region, there are at least three distinct sites of P element insertion at RpII215. Both 5' sites are within a noncoding portion of transcribed sequences. The sequences of four revertants of one P element insertion mutation (D50) indicate that the P element is either precisely deleted or internally deleted to restore RpII215 activity. Partial internal deletions of the P element result in different RpII215 activity levels, which appear to depend on the specific sequences that remain after excision.

Authors
Searles, LL; Greenleaf, AL; Kemp, WE; Voelker, RA
MLA Citation
Searles, LL, Greenleaf, AL, Kemp, WE, and Voelker, RA. "Sites of P element insertion and structures of P element deletions in the 5' region of Drosophila melanogaster RpII215." Mol Cell Biol 6.10 (October 1986): 3312-3319.
PMID
3025586
Source
pubmed
Published In
Molecular and Cellular Biology
Volume
6
Issue
10
Publish Date
1986
Start Page
3312
End Page
3319

Isolation of the nuclear gene encoding a subunit of the yeast mitochondrial RNA polymerase.

Antisera directed against the purified 145,000-dalton subunit of the Saccharomyces cerevisiae mitochondrial RNA polymerase have been used to immuno-screen a library of yeast genomic inserts constructed in the fusion protein expression vector, lambda gt11. A 4-kilobase pair yeast DNA fragment inserted into one of the recombinant bacteriophages appears to contain most or all of the gene that encodes the 145,000-dalton subunit.

Authors
Kelly, JL; Greenleaf, AL; Lehman, IR
MLA Citation
Kelly, JL, Greenleaf, AL, and Lehman, IR. "Isolation of the nuclear gene encoding a subunit of the yeast mitochondrial RNA polymerase." J Biol Chem 261.22 (August 5, 1986): 10348-10351.
PMID
2426263
Source
pubmed
Published In
The Journal of biological chemistry
Volume
261
Issue
22
Publish Date
1986
Start Page
10348
End Page
10351

Yeast RPO41 gene product is required for transcription and maintenance of the mitochondrial genome.

A 4-kilobase DNA fragment carried by a recombinant lambda gt11 bacteriophage appears to contain most of the coding information for the 145-kDa subunit of the Saccharomyces cerevisiae mitochondrial RNA polymerase. The RPO41 gene is located on chromosome VI, as determined by hybridization to electrophoretically separated yeast chromosomes. Hybridization and gene disruption/replacement experiments show that the RPO41 gene exists in a single copy and that its product is required for transcription and maintenance of the mitochondrial genome.

Authors
Greenleaf, AL; Kelly, JL; Lehman, IR
MLA Citation
Greenleaf, AL, Kelly, JL, and Lehman, IR. "Yeast RPO41 gene product is required for transcription and maintenance of the mitochondrial genome." Proc Natl Acad Sci U S A 83.10 (May 1986): 3391-3394.
PMID
3517858
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
83
Issue
10
Publish Date
1986
Start Page
3391
End Page
3394

Dye-ligand affinity chromatography of RNA polymerase II.

The binding of wheat germ RNA polymerase II to five different dye-ligand chromatography gels (Matrex gels, Amicon Corp.) was tested. A quantitative binding of the enzyme to four of the gels, namely Dyematrex gels Blue A, Blue B, Red A and Green A was observed. Only the Orange A gel column failed to bind the enzyme strongly. Nearly 100% of the activity could be recovered from the Green A column by elution with high salt concentration and high pH. Under these conditions only a part of the activity was eluted from the other three columns since the enzyme bound tightly. Enzyme activity could be removed from the columns by elution with nucleotide substrates, but the yield from the Blue A, Blue B and Red A columns was still low (7 to 42%). The Green A Matrex gel appeared to be useful for the purification and analysis of RNA polymerase.

Authors
Skripal, IG; Weeks, JR; Greenleaf, AL
MLA Citation
Skripal, IG, Weeks, JR, and Greenleaf, AL. "Dye-ligand affinity chromatography of RNA polymerase II." Acta Biochim Biophys Hung 21.3 (1986): 215-224.
PMID
3099522
Source
pubmed
Published In
Acta Biochimica et Biophysica Hungarica
Volume
21
Issue
3
Publish Date
1986
Start Page
215
End Page
224

A mutation in the largest subunit of RNA polymerase II alters RNA chain elongation in vitro.

An in vitro transcription system which utilized a semisynthetic DNA template (Kadesch, T. R., and Chamberlin, M. J. (1982) J. Biol. Chem. 257, 5286-5295) was developed and used to compare RNA chain elongation by wild type and mutant RNA polymerases II of Drosophila. With this template, all of the active polymerases rapidly initiated RNA chains at synthetic single-stranded sites at the ends of the DNA, and then entered a long (15 to 20 min) period of elongation through duplex regions of template before any measurable termination occurred. A comparison of wild type and mutant polymerase activities during this elongation phase indicated that a mutation to amanitin resistance reduces the rate at which the enzyme elongates transcripts. The reduced elongation rate of the mutant was associated with an altered substrate Km. Because the polymerase II mutation is in the largest enzyme subunit (Greenleaf, A. L. (1983) J. Biol. Chem. 258, 13403-13406), these results demonstrate a functional role for this subunit during RNA chain elongation.

Authors
Coulter, DE; Greenleaf, AL
MLA Citation
Coulter, DE, and Greenleaf, AL. "A mutation in the largest subunit of RNA polymerase II alters RNA chain elongation in vitro." J Biol Chem 260.24 (October 25, 1985): 13190-13198.
PMID
2414275
Source
pubmed
Published In
The Journal of biological chemistry
Volume
260
Issue
24
Publish Date
1985
Start Page
13190
End Page
13198

Structure of the eukaryotic transcription apparatus: features of the gene for the largest subunit of Drosophila RNA polymerase II.

The Drosophila melanogaster RpII215 locus encodes the largest subunit of RNA polymerase II. We have now mapped the 7 kb transcript of the locus and have determined that it contains four exons and three introns. By sequencing 2582 nucleotides from the promoter-proximal end of the RpII215 locus, we have precisely mapped the start site of transcription and the splice sites of the first intron. Segments of the amino acid sequence predicted by the only long open reading frame of the RpII215 gene transcript display striking homology with corresponding segments of the beta subunit of E. coli RNA polymerase.

Authors
Biggs, J; Searles, LL; Greenleaf, AL
MLA Citation
Biggs, J, Searles, LL, and Greenleaf, AL. "Structure of the eukaryotic transcription apparatus: features of the gene for the largest subunit of Drosophila RNA polymerase II." Cell 42.2 (September 1985): 611-621.
PMID
2992806
Source
pubmed
Published In
Cell
Volume
42
Issue
2
Publish Date
1985
Start Page
611
End Page
621

A MUTATION IN THE LARGEST SUBUNIT OF RNA POLYMERASE-II ALTERS RNA CHAIN ELONGATION INVITRO

Authors
COULTER, DE; GREENLEAF, AL
MLA Citation
COULTER, DE, and GREENLEAF, AL. "A MUTATION IN THE LARGEST SUBUNIT OF RNA POLYMERASE-II ALTERS RNA CHAIN ELONGATION INVITRO." JOURNAL OF BIOLOGICAL CHEMISTRY 260.24 (1985): 3190-3198.
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
260
Issue
24
Publish Date
1985
Start Page
3190
End Page
3198

Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes.

Three different regions of Saccharomyces cerevisiae DNA were identified by using as hybridization probe a fragment of Drosophila melanogaster DNA that encodes an RNA polymerase II (EC 2.7.7.6) polypeptide. Two of these regions have been molecularly cloned. Each contains a sequence related not only to the D. melanogaster DNA fragment that was used as a probe in its isolation but also to the immediately adjacent DNA fragment of the D. melanogaster RNA polymerase II gene. The two cloned S. cerevisiae DNA sequences are each the template for single transcripts in vivo, one of 5.9 kilobases and the other of 4.6 kilobases. In vitro translation of hybrid-selected cellular RNA indicated that the former locus encodes a protein of Mr 220,000, equal in size to the largest polypeptide subunit of S. cerevisiae RNA polymerase II. Disruption of either gene by targeted integration of URA3+ DNA demonstrated that each is single-copy and essential in a haploid genome. We suggest that these S. cerevisiae loci are members of a family of related genes encoding the largest subunit polypeptides of RNA polymerases I, II, and III.

Authors
Ingles, CJ; Himmelfarb, HJ; Shales, M; Greenleaf, AL; Friesen, JD
MLA Citation
Ingles, CJ, Himmelfarb, HJ, Shales, M, Greenleaf, AL, and Friesen, JD. "Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes." Proc Natl Acad Sci U S A 81.7 (April 1984): 2157-2161.
PMID
6326108
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
81
Issue
7
Publish Date
1984
Start Page
2157
End Page
2161

Frequent imprecise excision among reversions of a P element-caused lethal mutation in Drosophila

Authors
Voelker, RA; Greenleaf, AL; Gyurkovics, H
MLA Citation
Voelker, RA, Greenleaf, AL, and Gyurkovics, H. "Frequent imprecise excision among reversions of a P element-caused lethal mutation in Drosophila." Genetics 107.2 (1984): 279-294.
PMID
17246216
Source
scival
Published In
Genetics
Volume
107
Issue
2
Publish Date
1984
Start Page
279
End Page
294

Affinity-purified antibody as a probe of RNA polymerase II subunit structure

Authors
Robbins, A; Dynan, WS; Greenleaf, A; Tjian, R
MLA Citation
Robbins, A, Dynan, WS, Greenleaf, A, and Tjian, R. "Affinity-purified antibody as a probe of RNA polymerase II subunit structure." Journal of Molecular and Applied Genetics 2.4 (1984): 343-353.
PMID
6203994
Source
scival
Published In
Journal of Molecular and Applied Genetics
Volume
2
Issue
4
Publish Date
1984
Start Page
343
End Page
353

Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes

Three different regions of Saccharomyces cerevisiae DNA were identified by using as hybridization probe a fragment of Drosophila melanogaster DNA that encodes an RNA polymerase II (EC 2.7.7.6) polypeptide. Two of these regions have been molecularly cloned. Each contains a sequence related not only to the D. melanogaster DNA fragment that was used as a probe in its isolation but also to the immediately adjacent DNA fragment of the D. melanogaster RNA polymerase II gene. The two cloned S. cerevisiae DNA sequences are each the template for single transcripts in vivo, one of 5.9 kilobases and the other of 4.6 kilobases. In vitro translation of hybrid-selected cellular RNA that the former locus encodes a protein of M(r) 220,000, equal in size to the largest polypeptide subunit of S. cerevisiae RNA polymerase II. Disruption of either gene by targeted integration of URA3+ DNA demonstrated that each is single-copy and essential in a haploid genome. We suggest that these S. cerevisiae loci are members of a family of related genes encoding the largest subunit polypeptides of RNA polymerases I, II, and III.

Authors
Ingles, CJ; Himmelfarb, HJ; Shales, M; Greenleaf, AL; Friesen, JD
MLA Citation
Ingles, CJ, Himmelfarb, HJ, Shales, M, Greenleaf, AL, and Friesen, JD. "Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes." Proceedings of the National Academy of Sciences of the United States of America 81.7 I (1984): 2157-2161.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
81
Issue
7 I
Publish Date
1984
Start Page
2157
End Page
2161

Amanitin-resistant RNA polymerase II mutations are in the enzyme's largest subunit.

A fragment of the Drosophila melanogaster RpIIC4 locus, which encodes the RNA polymerase II subunit that determines amanitin sensitivity, was inserted into a bacterial plasmid cloning vehicle useful for over-production of hybrid proteins. Two plasmid constructions encoded hybrid proteins that reacted with antibodies against D. melanogaster RNA polymerase II. Use of subunit-specific antibodies indicated that these hybrid proteins displayed antigenic determinants unique to the largest polypeptide (215 kDa) of the enzyme. This RpII locus, the site at which mutations to amanitin-resistance occur, must therefore encode the largest polymerase II subunit.

Authors
Greenleaf, AL
MLA Citation
Greenleaf, AL. "Amanitin-resistant RNA polymerase II mutations are in the enzyme's largest subunit." J Biol Chem 258.22 (November 25, 1983): 13403-13406.
PMID
6417125
Source
pubmed
Published In
The Journal of biological chemistry
Volume
258
Issue
22
Publish Date
1983
Start Page
13403
End Page
13406

Identification of a structural gene for a RNA polymerase II polypeptide in Drosophila melanogaster and mammalian species.

Using subclones representing 14 kilobase pairs (kb) of DNA from the Drosophila melanogaster RNA polymerase II (EC 2.7.7.6) X-linked genetic locus, RpII, we have identified four poly(A)+ RNA transcripts in adult flies. The DNA encoding only one of these, a 7-kb transcript, cross-hybridized to mammalian DNA. DNA from alpha-amanitin-resistant (AmaR) Chinese hamster ovary (CHO) and human cells was used to transform the temperature-sensitive (TS) RNA polymerase II Syrian hamster mutant TsAF8. The acquisition of the TS+ AmaR RNA polymerase II phenotype was accompanied by the appearance of donor-DNA-specific restriction fragments that cross-hybridize to the D. melanogaster 7-kb transcript DNA. This D. melanogaster DNA and the related DNA detected in mammalian species must therefore be the structural gene for a RNA polymerase II polypeptide.

Authors
Ingles, CJ; Biggs, J; Wong, JK; Weeks, JR; Greenleaf, AL
MLA Citation
Ingles, CJ, Biggs, J, Wong, JK, Weeks, JR, and Greenleaf, AL. "Identification of a structural gene for a RNA polymerase II polypeptide in Drosophila melanogaster and mammalian species." Proc Natl Acad Sci U S A 80.11 (June 1983): 3396-3400.
PMID
6407013
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
80
Issue
11
Publish Date
1983
Start Page
3396
End Page
3400

α-Amanitin tolerance in mycophagous Drosophila

Six species of Drosophila were tested for tolerance to the mushroom toxin α-amanitin, a potent inhibitor of RNA polymerase II. Three nonmycophagous species - D. melanogaster, D. immigrans, and D. pseudoobscura - showed very low survival and long development times in the presence of amanitin. Three mycophagous species - D. putrida, D. recens, and D. tripunctata - showed little or no sensitivity. Analysis in vitro indicated that this tolerance is not based on alteration of the molecular structure of RNA polymerase II.

Authors
Jaenike, J; Grimaldi, DA; Sluder, AE; Greenleaf, AL
MLA Citation
Jaenike, J, Grimaldi, DA, Sluder, AE, and Greenleaf, AL. "α-Amanitin tolerance in mycophagous Drosophila." Science 221.4606 (1983): 165-167.
Source
scival
Published In
Science
Volume
221
Issue
4606
Publish Date
1983
Start Page
165
End Page
167

AMANITIN-RESISTANT RNA POLYMERASE-II MUTATIONS ARE IN THE ENZYMES LARGEST SUBUNIT

Authors
GREENLEAF, AL
MLA Citation
GREENLEAF, AL. "AMANITIN-RESISTANT RNA POLYMERASE-II MUTATIONS ARE IN THE ENZYMES LARGEST SUBUNIT." JOURNAL OF BIOLOGICAL CHEMISTRY 258.22 (1983): 3403-3406.
Source
wos-lite
Published In
The Journal of biological chemistry
Volume
258
Issue
22
Publish Date
1983
Start Page
3403
End Page
3406

ALPHA-AMANITIN TOLERANCE IN MYCOPHAGOUS DROSOPHILA

Authors
JAENIKE, J; GRIMALDI, DA; SLUDER, AE; GREENLEAF, AL
MLA Citation
JAENIKE, J, GRIMALDI, DA, SLUDER, AE, and GREENLEAF, AL. "ALPHA-AMANITIN TOLERANCE IN MYCOPHAGOUS DROSOPHILA." SCIENCE 221.4606 (1983): 165-167.
PMID
17769215
Source
wos-lite
Published In
Science
Volume
221
Issue
4606
Publish Date
1983
Start Page
165
End Page
167
DOI
10.1126/science.221.4606.165

A STRUCTURAL GENE FOR AN RNA POLYMERASE-II SUBUNIT

Authors
GREENLEAF, AL; BIGGS, J; JOKERST, RS; WEEKS, JR
MLA Citation
GREENLEAF, AL, BIGGS, J, JOKERST, RS, and WEEKS, JR. "A STRUCTURAL GENE FOR AN RNA POLYMERASE-II SUBUNIT." FEDERATION PROCEEDINGS 42.7 (1983): 1805-1805.
Source
wos-lite
Published In
The FASEB Journal
Volume
42
Issue
7
Publish Date
1983
Start Page
1805
End Page
1805

CLONING AND GENETIC-ANALYSIS OF HYBRID DYSGENESIS-INDUCED ALLELES AT AN RNA POLYMERASE-II LOCUS IN DROSOPHILA-MELANOGASTER

Authors
VOELKER, RA; HUANG, SM; GYURKOVICS, H; WISELY, GB; BINGHAM, PM; SEARLES, LL; JOKERST, RS; GREENLEAF, AL
MLA Citation
VOELKER, RA, HUANG, SM, GYURKOVICS, H, WISELY, GB, BINGHAM, PM, SEARLES, LL, JOKERST, RS, and GREENLEAF, AL. "CLONING AND GENETIC-ANALYSIS OF HYBRID DYSGENESIS-INDUCED ALLELES AT AN RNA POLYMERASE-II LOCUS IN DROSOPHILA-MELANOGASTER." ENVIRONMENTAL HEALTH PERSPECTIVES 52.OCT (1983): 285-285.
Source
wos-lite
Published In
Environmental health perspectives
Volume
52
Issue
OCT
Publish Date
1983
Start Page
285
End Page
285

Identification of a structural gene for a RNA polymerase II polypeptide in Drosophila melanogaster and mammalian species

Authors
Ingles, CJ; Biggs, J; Wong, JKC; Weeks, JR; Greenleaf, AL
MLA Citation
Ingles, CJ, Biggs, J, Wong, JKC, Weeks, JR, and Greenleaf, AL. "Identification of a structural gene for a RNA polymerase II polypeptide in Drosophila melanogaster and mammalian species." Proceedings of the National Academy of Sciences of the United States of America 80.11 I (1983): 3396-3400.
Source
scival
Published In
Proceedings of the National Academy of Sciences of USA
Volume
80
Issue
11 I
Publish Date
1983
Start Page
3396
End Page
3400

Molecular cloning of sequences from a Drosophila RNA polymerase II locus by P element transposon tagging.

We have identified a lethal mutation in the D. melanogaster RNA polymerase II locus, RpIIC4, caused by insertion of a transposable element associated with the phenomenon of hybrid dysgenesis (P element). Using previously cloned P element sequences as a hybridization probe we have isolated a hybrid lambda phage clone carrying a 10 kb genomic DNA fragment containing a 1.3 kb P element insert and flanking sequences from the RpII locus. The non-P sequences in this clone (lambda DmRpII-1) hybridize to polytene chromosome band region 10C, the cytogenetic location of RpIIC4, and revertants which lose the lethal RNA polymerase II mutation also lose P element sequences from the locus. We have generated several additional P element insertions into the locus and shown that they can occur at two or more different sites. These experiments illustrate that mutagenesis by P element insertion and use of cloned P DNA to retrieve the DNA sequences into which insertion has occurred may be a general method for cloning genetically defined loci in Drosophila.

Authors
Searles, LL; Jokerst, RS; Bingham, PM; Voelker, RA; Greenleaf, AL
MLA Citation
Searles, LL, Jokerst, RS, Bingham, PM, Voelker, RA, and Greenleaf, AL. "Molecular cloning of sequences from a Drosophila RNA polymerase II locus by P element transposon tagging." Cell 31.3 Pt 2 (December 1982): 585-592.
PMID
6297774
Source
pubmed
Published In
Cell
Volume
31
Issue
3 Pt 2
Publish Date
1982
Start Page
585
End Page
592

Immunological studies of RNA polymerase II using antibodies to subunits of Drosophila and wheat germ enzyme.

We induced goat antibodies to Drosophila RNA polymerase II and rabbit antibodies to the isolated 215,000-dalton and 140,000-dalton polymerase II subunits (P215 and P140, respectively). Similarly, we induced rabbit antibodies to wheat germ RNA polymerase II and to the 220,000-dalton subunit and 140,000-dalton subunit (P220 and P140, respectively). Anti-polymerase antibodies precipitated the homologous native enzyme and inhibited its activity in vitro, while several of the anti-subunit sera did neither. The anti-Drosophila P215 serum specifically labeled RNA polymerase II fixed in situ on polytene chromosomes. We reacted the antibodies with polymerase subunits separated by sodium dodecyl sulfate gel electrophoresis and electrophoretically transferred to nitrocellulose ("protein blotting"). Each antibody to whole polymerase reacted with multiple subunits, while the anti-subunit sera each reacted specifically with the subunit employed as immunogen. The anti-subunit sera also cross-reacted with the analogous subunit from several heterologous polymerases II (from yeast, wheat germ, Drosophila, and calf thymus), demonstrating shared subunit-specific determinants in polymerase II from widely divergent organisms. The anti-polymerase sera also showed cross-reactivity with subunits of heterologous enzymes, but only in one case did the cross-reactivity involve subunits other than the two largest ones. Specifically, the goat anti-Drosophila polymerase serum displayed easily detectable cross-reactivity with four low molecular weight subunits of calf thymus polymerase II, providing a unique demonstration of antigenic relatedness of small RNA polymerase II subunits from different higher eukaryotes.

Authors
Weeks, JR; Coulter, DE; Greenleaf, AL
MLA Citation
Weeks, JR, Coulter, DE, and Greenleaf, AL. "Immunological studies of RNA polymerase II using antibodies to subunits of Drosophila and wheat germ enzyme." J Biol Chem 257.10 (May 25, 1982): 5884-5892.
PMID
6175634
Source
pubmed
Published In
The Journal of biological chemistry
Volume
257
Issue
10
Publish Date
1982
Start Page
5884
End Page
5892

Properties of mutationally altered RNA polymerases II of Drosophila.

We tested and compared several in vitro properties of wild type and mutant RNA polymerases II from Drosophila melanogaster, using several different mutants of a single X-linked genetic locus, RpIIC4 (Greenleaf, A. L., Weeks, J. R., Voelker, R. A., Ohnishi, S., and Dickson, B. (1980) Cell 21, 785-792); the mutants tested included the original amanitin-resistant mutant, C4, which is nonconditional, plus the temperature-sensitive mutants A9, C20, E28, and 1Fb40. Using a tritium-labeled amanitin derivative, we demonstrated that C4 polymerase has a reduced binding affinity for amanitin. The C4 polymerase was as stable to thermal denaturation as the wild type enzyme, and the two enzymes had similar specific activities, ionic strength and Mn2+ requirements, and apparent Km values for UTP and GTP when assayed in the presence of Mn2+. However, with Mg2+ as the divalent cation, C4 polymerase was less active than wild type and had 2-fold higher apparent Km values for UTP and GTP. Three of the temperature-sensitive mutants, A9, C20, and E28, were derived from the amanitin-resistant mutant C4; the polymerase II activities from these mutants displayed resistance to alpha-amanitin in vitro identical with that of the C4 enzyme. C20, E28, and 1Fb40 polymerases were markedly less stable to thermal denaturation in vitro than wild type polymerase. The results presented indicate that the mutations at the RNA polymerase locus (RpIIC4-) directly alter the structure of the enzyme, providing conclusive evidence that the locus is a structural gene for a polymerase II subunit.

Authors
Coulter, DE; Greenleaf, AL
MLA Citation
Coulter, DE, and Greenleaf, AL. "Properties of mutationally altered RNA polymerases II of Drosophila." J Biol Chem 257.4 (February 25, 1982): 1945-1952.
PMID
6799516
Source
pubmed
Published In
The Journal of biological chemistry
Volume
257
Issue
4
Publish Date
1982
Start Page
1945
End Page
1952

MOLECULAR-CLONING OF SEQUENCES FROM A DROSOPHILA RNA POLYMERASE-II LOCUS BY P-ELEMENT TRANSPOSON TAGGING

Authors
SEARLES, LL; JOKERST, RS; BINGHAM, PM; VOELKER, RA; GREENLEAF, AL
MLA Citation
SEARLES, LL, JOKERST, RS, BINGHAM, PM, VOELKER, RA, and GREENLEAF, AL. "MOLECULAR-CLONING OF SEQUENCES FROM A DROSOPHILA RNA POLYMERASE-II LOCUS BY P-ELEMENT TRANSPOSON TAGGING." CELL 31.3 (1982): 585-592.
Source
wos-lite
Published In
Cell
Volume
31
Issue
3
Publish Date
1982
Start Page
585
End Page
592
DOI
10.1016/0092-8674(82)90314-2

Molecular cloning of sequences from a drosophila RNA polymerase II locus by P element transposon tagging

We have identified a lethal mutation in the D. melanogaster RNA polymerase II locus, RpllC4, caused by insertion of a transposable element associated with the phenomenon of hybrid dysgenesis (P element). Using previously cloned P element sequences as a hybridization probe we have isolated a hybrid lambda phage clone carrying a 10 kb genomic DNA fragment containing a 1.3 kb P element insert and flanking sequences from the Rpll locus. The non-P sequences in this clone (λDmRpll-1) hybridize to polytene chromosome band region 10C, the cytogenetic location of RpllC4, and revertants which lose the lethal RNA polymerase II mutation also lose P element sequences from the locus. We have generated several additional P element insertions into the locus and shown that they can occur at two or more different sites. These experiments illustrate that mutagenesis by P element insertion and use of cloned P DNA to retrieve the DNA sequences into which insertion has occurred may be a general method for cloning genetically defined loci in Drosophila. © 1982.

Authors
Searles, LL; Jokers, RS; Bingham, PM; Voelker, RA; Greenleaf, AL
MLA Citation
Searles, LL, Jokers, RS, Bingham, PM, Voelker, RA, and Greenleaf, AL. "Molecular cloning of sequences from a drosophila RNA polymerase II locus by P element transposon tagging." Cell 31.3 PART 2 (1982): 585-592.
Source
scival
Published In
Cell
Volume
31
Issue
3 PART 2
Publish Date
1982
Start Page
585
End Page
592

Genetic and biochemical characterization of mutants at an RNA polymerase II locus in D. melanogaster.

We previously described an alpha-amanitin-resistant mutant of D. melanogaster (AmaC4 or simply C4) with an altered, amanitin-resistant RNA polymerase II. We have now more fully characterized this mutant genetically and biochemically. We genetically mapped C4 to position 35.66 on the X chromosome and cytogenetically localized it to the polytene chromosome band interval 10C2-10D4. We then demonstrated that C4 is allelic to a previously known lethal-mutable locus I(1)L5 in this chromosomal region. Several known lethal alleles of L5 in fact affected the properties of RNA polymerase II in vitro. Following EMS mutagenesis of the C4-bearing chromosome we recovered new lethal L5 alleles, some of which were shown biochemically to have an altered amanitin-resistance polymerase II component. Furthermore, we induced mutants of C4 that had lost amanitin-resistance and showed that these mutants were also lethal alleles of L5. All the lethal alleles of L5 failed to completely complement each other genetically, and when analyzed biochemically their polymerase II displayed altered enzymatic properties. We conclude that C4 is an allele of the L5 locus and that this locus is most probably a structural gene for a subunit of RNA polymerase II. Some of the mutants at this locus display developmental abnormalities.

Authors
Greenleaf, AL; Weeks, JR; Voelker, RA; Ohnishi, S; Dickson, B
MLA Citation
Greenleaf, AL, Weeks, JR, Voelker, RA, Ohnishi, S, and Dickson, B. "Genetic and biochemical characterization of mutants at an RNA polymerase II locus in D. melanogaster." Cell 21.3 (October 1980): 785-792.
PMID
6777048
Source
pubmed
Published In
Cell
Volume
21
Issue
3
Publish Date
1980
Start Page
785
End Page
792

RNA POLYMERASE-II MUTANTS OF DROSOPHILA

Authors
GREENLEAF, AL; COULTER, D; WEEKS, J; VOELKER, RA
MLA Citation
GREENLEAF, AL, COULTER, D, WEEKS, J, and VOELKER, RA. "RNA POLYMERASE-II MUTANTS OF DROSOPHILA." FEDERATION PROCEEDINGS 39.6 (1980): 2111-2111.
Source
wos-lite
Published In
The FASEB Journal
Volume
39
Issue
6
Publish Date
1980
Start Page
2111
End Page
2111

Alpha-amanitin-resistant D. melanogaster with an altered RNA polymerase II.

Following EMS mutagenesis we recovered a mutant of D. melanogaster that grows at concentrations of alpha-amanitin lethal to wild-type. To our knowledge this mutant represents the first example of an amanitin-resistant eucaryotic organism. The amanitin resistance of the mutant (AmaC4) is due to an alteration in its DNA-dependent RNA polymerase II, which is approximately 250 times less sensitive to inhibition by amanitin than the wild-type polymerase II whether tested in nuclei, in partially-fractionated extracts or as a highly purified enzyme. While the wild-type enzyme activity is inhibited 50% by 2.1 x 10(-8) M alpha-amanitin, inhibition of 50% of the AmaC4 RNA polymerase II activity requires a toxin concentration of 5.6 x 10(-6) M. The mutation responsible for the amanitin resistance of AmaC4 is on the X chromosome near the vermillion locus.

Authors
Greenleaf, AL; Borsett, LM; Jiamachello, PF; Coulter, DE
MLA Citation
Greenleaf, AL, Borsett, LM, Jiamachello, PF, and Coulter, DE. "Alpha-amanitin-resistant D. melanogaster with an altered RNA polymerase II." Cell 18.3 (November 1979): 613-622.
PMID
117900
Source
pubmed
Published In
Cell
Volume
18
Issue
3
Publish Date
1979
Start Page
613
End Page
622

α-amanitin-resistant D. melanogaster with an altered RNA polymerase II

Following EMS mutagenesis we recovered a mutant of D. melanogaster that grows at concentrations of α-amanitin lethal to wild-type. To our knowledge this mutant represents the first example of an amanitin-resistant eucaryotic organism. The amanitin resistance of the mutant (AmaC4) is due to an alteration in its DNA-dependent RNA polymerase II, which is approximately 250 times less sensitive to inhibition by amanitin than the wild-type polymerase II whether tested in nuclei, in partially-fractionated extracts or as a highly purified enzyme. While the wild-type enzyme activity is inhibited 50% by 2.1 × 10-8 M α-amanitin, inhibition of 50% of the AmaC4 RNA polymerase II activity requires a toxin concentration of 5.6 × 10-6 M. The mutation responsible for the amanitin resistance of AmaC4 is on the X chromosome near the vermillion locus. © 1979.

Authors
Greenleaf, AL; Borsett, LM; Jiamachello, PF; Coulter, DE
MLA Citation
Greenleaf, AL, Borsett, LM, Jiamachello, PF, and Coulter, DE. "α-amanitin-resistant D. melanogaster with an altered RNA polymerase II." Cell 18.3 (1979): 613-622.
Source
scival
Published In
Cell
Volume
18
Issue
3
Publish Date
1979
Start Page
613
End Page
622

RNA polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes.

Using indirect immunofluorescence visualization techniques we investigated the distribution of RNA polymerase B (or II) and histone H1 at heat shock puff loci in Drosophila melanogaster polytene chromosomes at different times during and after heat shock. After heat treatments of from 5 to 45 min, the heat shock puff displayed intense fluorescence when stained for RNA polymerase B, but relatively little fluorescence when stained for histone H1. Returning heat shocked larvae to room temperature resulted in the appearance of a distinctive pattern of RNA polymerase-associated fluorescence in the heat shock puff at 87C, presumably reflecting events associated with the inactivation and regression of this puff. Large differences observed in the apparent RNA polymerase B content of puffs of similar size suggest that the interaction of RNA polymerase B with chromosomal loci does not depend on simply the state of condensation or decondensation of the chromatin.

Authors
Greenleaf, AL; Plagens, U; Jamrich, M; Bautz, EK
MLA Citation
Greenleaf, AL, Plagens, U, Jamrich, M, and Bautz, EK. "RNA polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes." Chromosoma 65.2 (January 16, 1978): 127-136.
PMID
414900
Source
pubmed
Published In
Chromosoma
Volume
65
Issue
2
Publish Date
1978
Start Page
127
End Page
136

Functional organization of polytene chromosomes.

Authors
Jamrich, M; Greenleaf, AL; Bautz, FA; Bautz, EK
MLA Citation
Jamrich, M, Greenleaf, AL, Bautz, FA, and Bautz, EK. "Functional organization of polytene chromosomes." Cold Spring Harb Symp Quant Biol 42 Pt 1 (1978): 389-396.
PMID
98281
Source
pubmed
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
42 Pt 1
Publish Date
1978
Start Page
389
End Page
396

Functional organization of polytene chromosomes

Authors
Jamrich, M; Greenleaf, AL; Bautz, FA; Bautz, EKF
MLA Citation
Jamrich, M, Greenleaf, AL, Bautz, FA, and Bautz, EKF. "Functional organization of polytene chromosomes." Cold Spring Harbor Symposia on Quantitative Biology 42.1 (December 1, 1977): 389-396.
Source
scopus
Published In
Cold Spring Harbor Laboratory: Symposia on Quantitative Biology
Volume
42
Issue
1
Publish Date
1977
Start Page
389
End Page
396

Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster.

RNA polymerase (RNA nucleotidyltransferase) B (or II) and histone H1 of Drosophila melanogster were localized on salivary gland polytene chromosomes using the indirect immunofluorescence technique. RNA polymerase B is present almost exclusively in puffs and interband regions, whereas histone H1 is found primarily in bands. The puff at region 3C, known to be transcriptionally active in larval salivary glands, gives a bright fluorescence with antibodies against RNA polymerase B. This fluorescence disappears after exposure of the larvae to 37 degrees for 45 min. The heat shock treatment results in a general reduction of fluorescence intensity with the appearance of brightly staining heat shock puffs. Heat-induced removal of RNA polymerase molecules from a puff does not immediately alter its morphology. We propose than an interband represents that fraction of the total number of gene copies in a band that are active, the inactive copies being present in a condensed form in the adjacent band. Large puffs would originate through the decondensation and activation of most or all gene copies in a band.

Authors
Jamrich, M; Greenleaf, AL; Bautz, EK
MLA Citation
Jamrich, M, Greenleaf, AL, and Bautz, EK. "Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster." Proc Natl Acad Sci U S A 74.5 (May 1977): 2079-2083.
PMID
405671
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
74
Issue
5
Publish Date
1977
Start Page
2079
End Page
2083

Distribution of RNA polymerase on Drosophila polytene chromosomes as studied by indirect immunofluorescence.

Using indirect immunofluorescence visualization techniques we investigated the in situ distribution of RNA polymerase B on Drosophila melanogaster polytene chromosomes. The enzyme was found at many sites distributed throughout the genome in a pattern clearly distinct from that observed for histone H1, but it was especially concentrated in puffs induced by heat shock.

Authors
Plagens, U; Greenleaf, AL; Bautz, EK
MLA Citation
Plagens, U, Greenleaf, AL, and Bautz, EK. "Distribution of RNA polymerase on Drosophila polytene chromosomes as studied by indirect immunofluorescence." Chromosoma 59.2 (December 16, 1976): 157-165.
PMID
827428
Source
pubmed
Published In
Chromosoma
Volume
59
Issue
2
Publish Date
1976
Start Page
157
End Page
165

In vitro proteolysis of a large subunit of Drosophila melanogaster RNA polymerase B.

Authors
Greenleaf, AL; Haars, R; Bautz, EK
MLA Citation
Greenleaf, AL, Haars, R, and Bautz, EK. "In vitro proteolysis of a large subunit of Drosophila melanogaster RNA polymerase B." FEBS Lett 71.2 (December 1, 1976): 205-208.
PMID
826408
Source
pubmed
Published In
FEBS Letters
Volume
71
Issue
2
Publish Date
1976
Start Page
205
End Page
208

RNA polymerase from sporulating Bacillus subtilis. Purification and properties of a modified form of the enzyme containing two sporulation polypeptides.

A new form of DNA-dependent RNA polymerase termed enzyme III has been purified from sporulating cells of Bacillus subtilis. In addition to the subunits of core RNA polymerase (beta', beta, alpha, and omega), enzyme III contains sporulation-specific polypeptides of 85,000 (P85) and 27,000 (P27) daltons. P85 corresponds to an RNA polymerase-binding protein previously identified by precipitation of RNA polymerase from crude extracts of sporulating cells with antibody directed against core enzyme. Both P85 and P27 co-purified with RNA polymerase highly purified by gel filtration, DEAE-cellulose chromatography, phosphocellulose chromatography, and glycerol gradient centrifugation. Enzyme III bound more tightly to phosphocellulose and sedimented more rapidly during zone centrifugation than did RNA polymerase lacking the sporulation polypeptides. RNA polymerase containing P85 and P27 transcribed B. subtilis DNA about 4.5 times more actively than did core RNA polymerase, although both enzymes exhibited similar activities with poly(dA-dT) and phage phie DNA as templates. Enzyme III and core RNA polymerase also differed in their response to increasing concentrations of Mg2+ and KCl.

Authors
Linn, T; Greenleaf, AL; Losick, R
MLA Citation
Linn, T, Greenleaf, AL, and Losick, R. "RNA polymerase from sporulating Bacillus subtilis. Purification and properties of a modified form of the enzyme containing two sporulation polypeptides." J Biol Chem 250.24 (December 25, 1975): 9256-9261.
PMID
811662
Source
pubmed
Published In
The Journal of biological chemistry
Volume
250
Issue
24
Publish Date
1975
Start Page
9256
End Page
9261

RNA polymerase B from Drosophila melanogaster larvae. Purification and partial characterization.

A purification procedure is described by which we obtained DNA-dependent RNA polymerase B (or II) from third-instar larvae of Drosophila melanogaster in essentially pure form. The enzyme is similar to the analogous polymerases from other eukaryotes in its enzymic and structural properties. It preferentially transcribes DNAs containing single-stranded regions, and it is inhibited by low amounts of the toxin alpha-amanitin; 50% inhibition occurs at an alpha-amanitin concentration of 0.03 mug/ml. Dodecylsulfate-polyacrylamide gel electrophoresis resolves the purified Drosophila polymerase B into ten polypeptides with molecular weights as follows: 1 (174000), 2 (137000), 3 (34000), 4 (22000), 5 (18000), 6 and 7 (16000), 8 (15000), and 9 and 10 (less than 15000). The relative amounts of polypeptides 1-4 were constant at molar ratios of approximately 1:1:1:2 in different preparations of the enzyme, while the amounts of polypeptides 5-10 showed more variation. An antiserum directed against the Drosophila RNA polymerase B inhibited the activity in vitro of the B enzymes from Drosophila, yeast, and calf thymus. However, only the Drosophila enzyme gave a precipitin reaction with the antiserum. When the antiserum was added to Drosophila RNA polymerase B at different stages of the purification, the resulting precipitates were found to contain nearly constant proportions of seven of the ten polypeptides present in the purified enzyme.

Authors
Greenleaf, AL; Bautz, EK
MLA Citation
Greenleaf, AL, and Bautz, EK. "RNA polymerase B from Drosophila melanogaster larvae. Purification and partial characterization." Eur J Biochem 60.1 (December 1, 1975): 169-179.
PMID
812697
Source
pubmed
Published In
European journal of biochemistry / FEBS
Volume
60
Issue
1
Publish Date
1975
Start Page
169
End Page
179

Appearance of a ribonucleic acid polymerase-binding protein in asporogenous mutants of Bacillus subtilis.

A 70,000-dalton protein that is found in sporulating Bacillus subtilis and that binds to ribonucleic acid polymerase is present in asporogenous mutants that proceed to or beyond stage II of sporulation, but is absent from mutants blocked at stage zero.

Authors
Greenleaf, AL; Losick, R
MLA Citation
Greenleaf, AL, and Losick, R. "Appearance of a ribonucleic acid polymerase-binding protein in asporogenous mutants of Bacillus subtilis." J Bacteriol 116.1 (October 1973): 290-294.
PMID
4200841
Source
pubmed
Published In
Journal of bacteriology
Volume
116
Issue
1
Publish Date
1973
Start Page
290
End Page
294

Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation.

The activity of the sigma subunit of the RNA polymerase of Bacillus subtilis decreases markedly during the first 2 hr of sporulation. Moreover, sigma activity remains deficient throughout the sporulation process and in dormant spores. The time course of changes in RNA polymerase during sporulation indicates that alterations in the core of RNA polymerase occur after the loss of sigma activity. Core RNA polymerase purified after the second and before the ninth hour of sporulation fails to respond to vegetative sigma subunit in vitro and contains variable amounts of a 110,000-dalton polypeptide in place of the beta' subunit. Core RNA polymerase purified from dormant spores has a subunit structure indistinguishable from vegetative core enzyme.RNA polymerase purified by antibody precipitation from an extract of a mixture of sporulating and excess vegetative cells separately labeled with two different radioisotopes contains beta' subunit and no 110,000-dalton polypeptide. However, RNA polymerase purified from sporulating bacteria in the absence of excess vegetative cells progressively loses the beta' subunit at each stage of purification even in the presence of the protease inhibitor, phenylmethyl sulfonyl fluoride. These findings suggest that the alteration of the beta' subunit is due to proteolysis in vitro.

Authors
Linn, TG; Greenleaf, AL; Shorenstein, RG; Losick, R
MLA Citation
Linn, TG, Greenleaf, AL, Shorenstein, RG, and Losick, R. "Loss of the sigma activity of RNA polymerase of Bacillus subtilis during sporulation." Proc Natl Acad Sci U S A 70.6 (June 1973): 1865-1869.
PMID
4198276
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
70
Issue
6
Publish Date
1973
Start Page
1865
End Page
1869

Isolation of a new RNA polymerase-binding protein from sporulating Bacillus subtilis.

RNA polymerase was precipitated from extracts of radioactively labeled vegetative and sporulating Bacillus subtilis with antiserum prepared against vegetative core polymerase. The precipitates were solubilized and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antiserum added to an extract of vegetative B. subtilis precipitated only the known subunits of core RNA polymerase, but antiserum added to an extract of sporulating cells precipitated a new polypeptide of 70,000 daltons in addition to the subunits of core enzyme. The 70,000-dalton polypeptide precipitated from an extract of a mixture of vegetative and sporulating B. subtilis, separately labeled with two different radioisotopes, contained only the radioisotope characteristic of the sporulating cells. The 70,000-dalton protein has been freed of core RNA polymerase and extensively purified by chromatography on phosphocellulose. Precipitation of the purified 70,000-dalton protein by the anti-polymerase serum requires the prior addition of vegetative or sporulation core RNA polymerase. The reaction is specific since the purified protein is not precipitated during antibody precipitation of either phage lambda repressor or bovine serum albumin. The RNA polymerase-binding protein appears during the third hour of sporulation and is apparently not synthesized by the sporulation-defective mutant rfr 10.

Authors
Greenleaf, AL; Linn, TG; Losick, R
MLA Citation
Greenleaf, AL, Linn, TG, and Losick, R. "Isolation of a new RNA polymerase-binding protein from sporulating Bacillus subtilis." Proc Natl Acad Sci U S A 70.2 (February 1973): 490-494.
PMID
4631355
Source
pubmed
Published In
Proceedings of the National Academy of Sciences of USA
Volume
70
Issue
2
Publish Date
1973
Start Page
490
End Page
494

On the specificity of creatine kinase. New glycocyamines and glycocyamine analogs related to creatine.

Authors
Rowley, GL; Greenleaf, AL; Kenyon, GL
MLA Citation
Rowley, GL, Greenleaf, AL, and Kenyon, GL. "On the specificity of creatine kinase. New glycocyamines and glycocyamine analogs related to creatine." J Am Chem Soc 93.12 (October 20, 1971): 5542-5551.
PMID
5165680
Source
pubmed
Published In
Journal of the American Chemical Society
Volume
93
Issue
12
Publish Date
1971
Start Page
5542
End Page
5551

On the specificity of creatine kinase. New glycocyamines and glycocyamine analogs related to creatine

The specificity of rabbit muscle creatine kinase (adenosine triphosphate-creatine phosphotransferase, EC 2.7.3.2) for a series of new synthetic analogs of creatine has been investigated. Two of these analogs, 1-carboxymethyl-2-iminoimidazolidine and N-methyl-N-amidinoaminomethylphosphinic acid, are more reactive (31 and 13% as reactive as creatine, respectively) as substrates in the creatine kinase reaction than any analogs of creatine reported to date. New synthetic routes to substituted glycocyamines have been developed as well as some improvements made on existing synthetic procedures. Earlier synthetic difficulties are discussed in terms of solubility properties of the glycocyamines and their ease of cyclization to glycocyamidines. In the presence of the enzyme, adenosine triphosphate has been shown to phosphorylate the highly reactive analog 1-carboxymethyl-2-iminoimidazolidine on the primary amino group to give 1-carboxymethyl-2-(phosphonoimino)imidazolidine. This result and the other specificity results are discussed in terms of bulk tolerance and geometrical requirements at the active site of the enzyme for optimal activity of the creatine analogs.

Authors
Rowley, GL; Greenleaf, AL; Kenyon, GL
MLA Citation
Rowley, GL, Greenleaf, AL, and Kenyon, GL. "On the specificity of creatine kinase. New glycocyamines and glycocyamine analogs related to creatine." Journal of the American Chemical Society 93.21 (1971): 5542-5551.
Source
scival
Published In
Journal of the American Chemical Society
Volume
93
Issue
21
Publish Date
1971
Start Page
5542
End Page
5551
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