Modulation of RNA Polymerase II Elongation Efficiency by C-terminal Heptapeptide Repeat Domain Kinase I

Kim, Jae Pil; Greenleaf, Arno L.

doi:10.1074/jbc.272.17.10990

Cited by 89 publications

(64 citation statements)

References 41 publications

(42 reference statements)

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“…As a consequence, overexpression of cell cycle cyclins usually generates growth defects (9,60,68). The CTDK-I complex is implicated not in cell cycle regulation but rather in transcription regulation (26,31). Similarly to what has been observed for other C-type cyclins implicated in transcription control, CTK2 mRNA levels do not fluctuate during the cell cycle (data not shown).…”

Section: Resultsmentioning

confidence: 52%

“…CTDK-I is related to the Srb10/11 kinase complex, although it seems to display a distinct role in transcriptional control (26). In vitro studies carried out with HeLa nuclear extracts have shown that CTDK-I can modulate the elongation efficiency of RNA polymerase II (31). In humans, the closest kinase to Ctk1p is Cdk9p, a CTD kinase that was first described as a positive transcription elongation factor (38).…”

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confidence: 99%

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The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

Hautbergue

Goguel

1999

Molecular and Cellular Biology

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The yeast CTDK-I complex has been implicated in phosphorylation of the carboxy-terminal domain of the RNA polymerase II and in transcription control. It is composed of three polypeptides: Ctk1p and Ctk2p, a cyclin-dependent kinase and a C-type cyclin subunit, respectively; and Ctk3p, a third subunit of unknown function. Cyclins are regulatory proteins whose expression is tightly controlled at the protein level. In this study, we examined the regulation of Ctk2p expression in vivo. Surprisingly, unlike what has been described for cell cycle cyclins, steady-state levels of Ctk2p are composed of two relatively abundant forms, one of them phosphorylated. We show that this phosphorylated form is extremely unstable (half-life, 5 min) and that rapid proteolysis of Ctk2p exhibits growth-related regulation. Furthermore, our data establish that similar to the case for other naturally short-lived proteins, Ctk2p degradation is mediated by the ubiquitin-proteasome pathway. This is the first demonstration that a C-type cyclin is phosphorylated and targeted to the proteasome. Strikingly, neither phosphorylation nor destruction of Ctk2p requires its associated kinase Ctk1p, a feature fundamentally different from that which has been observed for cell cycle cyclins.Cyclin-dependent protein kinases (CDKs) were first identified as central regulators of the major transitions of the eukaryotic cell division cycle. Their activity is determined by cyclin binding, by both positive and negative regulatory phosphorylation, and by polypeptide CDK inhibitors (40, 41). A single cyclin-dependent kinase subunit associates sequentially with multiple cyclin partners whose abundance fluctuates during the cell cycle (43). Indeed, one aspect of CDK regulation is triggered by the rapid destruction of their cyclin partners (23). Several studies both in yeast and in mammals have demonstrated that cyclins are selectively degraded by the ubiquitindependent proteasome pathway (9,16,53,67). Substrates are first marked for degradation by conjugation with several molecules of ubiquitin, a highly conserved 76-amino-acid-long polypeptide that is joined reversibly by a covalent linkage.

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Section: Resultsmentioning

confidence: 52%

mentioning

confidence: 99%

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

Hautbergue

Goguel

1999

Molecular and Cellular Biology

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“…The kinase we used, CTDK-I (34 -36), is homologous to human P-TEFb (45) and to other biochemically uncharacterized human open reading frames (46). In view of the sequence relatedness and because CTDK-I and P-TEFb are both involved in phosphorylating RNAP II for elongation (47,48), the pattern of phosphates on our probe is expected to be very similar to that generated on the consensus repeats of the human CTD by P-TEFb or other elongation-related CTD kinases. Current in vivo data suggest that elongating RNAP II is predominantly phosphorylated on Ser-2 of the heptad repeats (49 -51).…”

Section: The Approachmentioning

confidence: 99%

Hyperphosphorylated C-terminal Repeat Domain-associating Proteins in the Nuclear Proteome Link Transcription to DNA/Chromatin Modification and RNA Processing

Carty

Greenleaf

2002

Molecular & Cellular Proteomics

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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. Molecular & Cellular Proteomics 1:598 -610, 2002.The C-terminal repeat domain (CTD), 1 a non-catalytic "tail" on the largest subunit of RNA polymerase, is a unique structure made up of as many as 52 repeats of the consensus heptamer YSPTSPS (1). That each seven-residue repeat contains five hydroxyl-containing side chains suggests that the CTD could be a good target for phosphorylation, and indeed, in elongating RNAP II the CTD is hyperphosphorylated (2-4); in preinitiating RNAP II, on the other hand, the CTD is not phosphorylated (e.g. see Ref. 5). The amino acid composition of the CTD also renders it very hydrophilic, and it is largely unstructured in aqueous solution (6, 7); in mammals a fully stretched out CTD could potentially extend some 1200 Å away from the globular body of the polymerase (whose diameter is ϳ150 Å, Ref. 8) (see e.g. Ref. 9). Its unusual properties render the CTD well suited to function as an extended scaffold to which transcription factors and other proteins could bind; indeed, a growing body of data indicates that such a scaffolding role is the principal function of the CTD. Experiments focusing on the hyperphosphorylated CTD characteristic of elongating RNAP II have revealed that a number of RNA processing factors bind to the phosphoCTD (e.g. see Refs. 10 and 11). Binding to the phosphoCTD tethers such factors to the transcription machinery where they can carry out their functions more efficiently (e.g. see Refs. 12-16); furthermore, certain factors or processes are stimulated by phosphoCTD binding (e.g. see . In addition, a pool of nontranscribing RNAP II carries a phosphoCTD and is associated with certain transcription and processing factors in potential assembly ar...

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“…Although both have been shown to assemble into preinitiation complexes (PICs) at the promoter, recent experiments utilizing analogue-sensitive mutants of both kinases suggest different, but partially overlapping roles for both enzymes in promoting transcription (12). The other two CTD kinases, Ctk1 in CTDK-I and Sgv1/Bur1, have been associated primarily with the elongation phase of transcription (13)(14)(15)(16)(17). Biochemical studies indicate that CTDK-I can stimulate elongation (17), and in vivo experiments show that elongating Pol II is aberrantly phosphorylated in ctk1 mutants (16,18).…”

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Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

2004

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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.The carboxyl-terminal repeat domain (CTD) 1 of the largest subunit of eukaryotic RNA polymerase II (Pol II) comprises tandem repeats of the consensus heptapeptide YSPTSPS. This highly conserved sequence, which is repeated 26 times in yeast and 52 times in mammals, is essential for viability. Phosphorylation of the CTD regulates the activities of the polymerase: only Pol II with an unphosphorylated CTD (Pol IIA) is thought to be able to assemble into preinitiation complexes at the promoter, whereas elongating polymerase has a hyperphosphorylated CTD (Pol IIO) (1,2). The serines at positions 2 and 5 within the heptad repeat represent major sites of phosphorylation during transcription. † Supported by National Institutes of Health Grant GM40505.© 2004 American Chemical Society * To whom correspondence should be addressed. . E-mail: arno@biochem.duke.edu. 1 Abbreviations: CTDK-I, CTD kinase I; Pol II, RNA polymerase II; CTD, C-terminal repeat domain; phosphoCTD, hyperphosphorylated CTD; PCTD, phosphoCTD; PCAP, phosphoCTD-associating protein; PCID, phosphoCTD-interacting domain; SGD, Saccharomyces Genome Database; SPR, surface plasmon resonance; RU, response units. NIH Publi...

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Modulation of RNA Polymerase II Elongation Efficiency by C-terminal Heptapeptide Repeat Domain Kinase I

Cited by 89 publications

References 41 publications

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

The Yeast C-Type Cyclin Ctk2p Is Phosphorylated and Rapidly Degraded by the Ubiquitin-Proteasome Pathway

Hyperphosphorylated C-terminal Repeat Domain-associating Proteins in the Nuclear Proteome Link Transcription to DNA/Chromatin Modification and RNA Processing

Expanding the Functional Repertoire of CTD Kinase I and RNA Polymerase II: Novel PhosphoCTD-Associating Proteins in the Yeast Proteome

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