An Unusual Eukaryotic Protein Phosphatase Required for Transcription by RNA Polymerase II and CTD Dephosphorylation in S. cerevisiae

Kobor, Michæl S.; Archambault, Jacques; Lester, William C.; Holstege, Frank C.P.; Gileadi, O.; Jansma, David B.; Jennings, Ezra G.; Kouyoumdjian, Fiona G.; Davidson, Alan R.; Young, Richard A.; Greenblatt, Jack

doi:10.1016/s1097-2765(00)80187-2

Cited by 186 publications

(239 citation statements)

References 30 publications

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“…The phosphatase Fcp1p dephosphorylates the C-terminal domain (CTD) of Pol II and is generally required for Pol II transcription in yeast (31,32). It was reported that fcp1-ts mutants had defects in Pol I transcription, and Fcp1p stimulated transcription by Pol I in vitro in specific and nonspecific transcription assays, supporting a role for Fcp1p in Pol I elongation (33).…”

Section: Discussionmentioning

confidence: 99%

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

Schneider

French

Osheim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

105

143

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Previous investigations into the mechanisms that control RNA Polymerase (Pol) I transcription have primarily focused on the process of transcription initiation, thus little is known regarding postinitiation steps in the transcription cycle. Spt4p and Spt5p are conserved throughout eukaryotes, and they affect elongation by Pol II. We have found that these two proteins copurify with Pol I and associate with the rDNA in vivo. Disruption of the gene for Spt4p resulted in a modest decrease in growth and rRNA synthesis rates at the permissive temperature, 30°C. Furthermore, biochemical and EM analyses showed clear defects in rRNA processing. These data suggest that Spt4p, Spt5p, and, potentially, other regulators of Pol I transcription elongation play important roles in coupling rRNA transcription to its processing and ribosome assembly.yeast Saccharomyces cerevisiae T he synthesis of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I is an important step in the synthesis of ribosomes, and its regulation is closely linked to the nutrient conditions and growth potential for the cell. Previous studies have identified essential components of the Pol I transcription apparatus as well as important cis-elements in Pol I promoters and revealed some potential mechanisms for regulation of transcription initiation (for reviews, see refs. 1-5). Unlike Pol II, however, little is known regarding mechanisms that regulate postinitiation steps of transcription by Pol I.One well characterized Pol II transcription elongation factor in yeast is a complex of two proteins, Spt4p and Spt5p (the complex will be referred to as Spt4͞5 here). The SPT4 and SPT5 genes were among many genes isolated for their ability to suppress transcription defects caused by insertions of the retrotransposon Ty1 (or the Ty1 long terminal repeats, ␦) in the 5Ј noncoding regions of yeast genes (6). Swanson and Winston (7) later showed that Spt4͞5 associates with Spt6p, which affects Pol II elongation through chromatin. However, Spt4p and Spt5p also form a separate complex, devoid of Spt6p, that associates with Pol II physically and genetically, and this interaction is important for transcription elongation (8). More recent work has shown that deletion of the nonessential gene SPT4 results in reduced efficiency of Pol II elongation through GC-rich DNA sequences (9) and a general decrease in Pol II processivity (10). Taken together, all of these data clearly support a role for Spt4͞5 in transcription elongation by Pol II in yeast.Spt4͞5 also plays a role in Pol II transcription elongation in mammalian cells. The mammalian homologues of Spt4p and Spt5p form a complex called the 5,6-dichloro-1-␤-Dribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF), which was originally identified as a factor that induces a DRB-dependent arrest of elongating Pol II complexes in reconstituted in vitro transcription assays (11). It was demonstrated that under nucleotide-limiting conditions, DSIF could also increase the rate of elongation of Pol II in vitro. Thus, work in mam...

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Section: Discussionmentioning

confidence: 99%

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

Schneider

French

Osheim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

105

143

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“…does not display any homology with other known protein phosphatases but possesses an unusual catalytic site present in a subset of phosphotransferases (13,14). FCP1 is a highly specific phosphatase as the CTD within a functional RNAP II remains its only known protein substrate.…”

Section: Rna Polymerase II (Rnap Ii)mentioning

confidence: 99%

“…p-Nitrophenyl phosphate has been used as an artificial substrate for Saccharomyces cerevisiae or Schizosaccharomyces pombe FCP1 (14,30). Indeed, p-nitrophenol was generated in proportion to the amounts of FCP1 in the assay (Fig.…”

Section: Ck2 Is the Major Fcp1 Kinase Present In Eggmentioning

confidence: 99%

FCP1 Phosphorylation by Casein Kinase 2 Enhances Binding to TFIIF and RNA Polymerase II Carboxyl-terminal Domain Phosphatase Activity

Palancade

Dubois

Bensaude

2002

Journal of Biological Chemistry

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Dephosphorylation of RNA polymerase II carboxylterminal domain (CTD) is required to resume sequential transcription cycles. FCP1 (TFIIF-dependent CTD phosphatase 1) is the only known phosphatase targeting RNAP II CTD. Here we show that in Xenopus laevis cells, xFCP1 is a phosphoprotein. On the basis of biochemical fractionation and drug sensitivity, casein kinase 2 (CK2) is shown to be the major kinase involved in xFCP1 phosphorylation in X. laevis egg extracts. CK2 phosphorylates xFCP1 mainly at a cluster of serines centered on Ser 457 . CK2-dependent phosphorylation enhances 4-fold the CTD phosphatase activity of FCP1 and its binding to the RAP74 subunit of general transcription factor TFIIF. These findings unravel a new mechanism regulating CTD phosphorylation and hence class II gene transcription.RNA polymerase II (RNAP II) 1 is a multiprotein complex in charge of eucaryotic mRNA synthesis (1). Rpb1, the largest RNAP II subunit, is extensively phosphorylatable on its carboxyl-terminal domain (CTD), which consists of up to 52 repeats of the consensus heptapeptide Tyr-Ser-Pro-Thr-Ser-ProSer (2). Two phosphoisoforms of RNAP II coexist in somatic cells in a dynamic equilibrium (3). The hypophosphorylated IIA form assembles into preinitiation complexes, whereas the hyperphosphorylated IIO form elongates mRNA synthesis and recruits mRNA processing factors (4). The conversion from IIA to IIO occurs during initiation and elongation of transcription and involves CTD kinases such as CDK7 and CDK9, respectively, included in the transcription factors TFIIH and p-TEFb (5, 6). CTD dephosphorylation is required to recycle RNAP II after a transcriptional round (7).In contrast to the numerous CTD kinases identified, only one CTD phosphatase has been characterized so far. It has been purified from yeast and mammalian cells (8,9), and the corresponding cDNA has been cloned by a two-hybrid screen using the RAP74 subunit of the general transcription factor TFIIF as a bait (10). FCP1 (TFIIF-dependent CTD Phosphatase 1) consists of a single 150-kDa polypeptide subunit (11, 12). FCP1does not display any homology with other known protein phosphatases but possesses an unusual catalytic site present in a subset of phosphotransferases (13,14). FCP1 is a highly specific phosphatase as the CTD within a functional RNAP II remains its only known protein substrate. FCP1 is recruited to RNAP II through docking sites on the Rpb4 subunit of RNAP II and on the RAP74 subunit of TFIIF (15, 16).FCP1 activity is regulated at various levels. First, competitive binding of general transcription factors TFIIF and TFIIB to FCP1 carboxyl-terminal domain, respectively, stimulates or inhibits its phosphatase activity (15,17). Second, free RNAP IIO molecules are better substrates than molecules engaged in transcription elongation complexes (18,19). Third, inhibition of FCP1 phosphatase activity by the human immunodeficiency viral protein Tat may contribute to stimulate transcriptional elongation from the human immunodeficiency virus promoter (20, 21).We had...

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“…In contrast to the four kinases, there are two known CTD phosphatases in yeast, . Biochemical as well as genetic evidence indicates that both proteins may function as CTD phosphatases in the context of transcription (16,19,20,(25)(26)(27)(28)(29)(30)(31).It is the interplay between these CTD kinases and phosphatases that influences which factors associate with transcribing polymerase and when during the transcription cycle such associations occur. As mentioned, the recruitment of capping enzyme early in the transcription cycle is believed to be one consequence of CTD phosphorylation on Ser5 by Kin28 (6,7).…”

mentioning

confidence: 99%

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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An Unusual Eukaryotic Protein Phosphatase Required for Transcription by RNA Polymerase II and CTD Dephosphorylation in S. cerevisiae

Cited by 186 publications

References 30 publications

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

RNA polymerase II elongation factors Spt4p and Spt5p play roles in transcription elongation by RNA polymerase I and rRNA processing

FCP1 Phosphorylation by Casein Kinase 2 Enhances Binding to TFIIF and RNA Polymerase II Carboxyl-terminal Domain Phosphatase Activity

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

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