Characterization of the Residues Phosphorylated in Vitro by Different C-terminal Domain Kinases

Trigon, Sylviane; Serizawa, Hiroaki; Conaway, Joan Weliky; Conaway, Ronald C.; Jackson, Stephen; Morange, Michel

doi:10.1074/jbc.273.12.6769

Cited by 108 publications

(114 citation statements)

References 67 publications

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“…Reduced CTD phosphorylation has also been observed in embryos depleted for ttb-1, the C. elegans TFIIB homolog, and is consistent with a general defect in RNAPII transcription (24). In vitro, CDK7 possesses robust CTD-kinase activity specific for phosphorylation at Ser-5 of the CTD consensus repeat (42)(43)(44)(45). Interestingly, using the monoclonal antibodies H5 and H14 (30,31), we found that phosphorylation at both Ser-2 and Ser-5 is reduced in cdk-7 mutants.…”

Section: Discussionmentioning

confidence: 84%

cdk-7 is required for mRNA transcription and cell cycle progression in Caenorhabditis elegans embryos

Wallenfang

Seydoux

2002

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

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CDK7 is a cyclin-dependent kinase proposed to function in two essential cellular processes: transcription and cell cycle regulation. CDK7 is the kinase subunit of the general transcription factor TFIIH that phosphorylates the C-terminal domain (CTD) of RNA polymerase II, and has been shown to be broadly required for transcription in Saccharomyces cerevisiae. CDK7 can also phosphorylate CDKs that promote cell cycle progression, and has been shown to function as a CDK-activating kinase (CAK) in Schizosaccharomyces pombe and Drosophila melanogaster. That CDK7 performs both functions in metazoans has been difficult to prove because transcription is essential for cell cycle progression in most cells. We have isolated a temperature-sensitive mutation in Caenorhabditis elegans cdk-7 and have used it to analyze the role of cdk-7 in embryonic blastomeres, where cell cycle progression is independent of transcription. Partial loss of cdk-7 activity leads to a general decrease in CTD phosphorylation and embryonic transcription, and severe loss of cdk-7 activity blocks all cell divisions. Our results support a dual role for metazoan CDK7 as a broadly required CTD kinase, and as a CAK essential for cell cycle progression.

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Section: Discussionmentioning

confidence: 84%

cdk-7 is required for mRNA transcription and cell cycle progression in Caenorhabditis elegans embryos

Wallenfang

Seydoux

2002

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

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“…Recently, CTD phosphorylation sites have been reported from studies using a GST-CTD fusion protein, CTD heptapeptide repeat peptides, and a free form of intact Pol II; for example, TFIIH phosphorylates Ser-5, and Cdk8-cyclin C of the SRB-and Med-containing complex NAT phosphorylates Ser-2 and Ser-5 (58,64). Since TFIIH is the only CTD kinase to exist in the reconstituted active PIC at transcription, we thought that phosphorylation of the CTD heptapeptide repeats (YSPTSPS; the first Tyr [Y] is here designated Tyr-1) of Pol II in the active PIC must correspond to the conformational change of Pol II to be processive.…”

Section: Resultsmentioning

confidence: 99%

Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

Yamamoto

Watanabe

Spek

et al. 2001

Molecular and Cellular Biology

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The general transcription factor TFIIE plays important roles in transcription initiation and in the transition to elongation. However, little is known about its function during these steps. Here we demonstrate for the first time that TFIIH-mediated phosphorylation of RNA polymerase II (Pol II) is essential for the transition to elongation. This phosphorylation occurs at serine position 5 (Ser-5) of the carboxy-terminal domain (CTD) heptapeptide sequence of the largest subunit of Pol II. In a human in vitro transcription system with a supercoiled template, this process was studied using a human TFIIE (hTFIIE) homolog from Caenorhabditis elegans (ceTFIIE␣ and ceTFIIE␤). ceTFIIE␤ could partially replace hTFIIE␤, whereas ceTFIIE␣ could not replace hTFIIE␣. We present the studies of TFIIE binding to general transcription factors and the effects of subunit substitution on CTD phosphorylation. As a result, ceTFIIE␣ did not bind tightly to hTFIIE␤, and ceTFIIE␤ showed a similar profile for binding to its human counterpart and supported an intermediate level of CTD phosphorylation. Using antibodies against phosphorylated serine at either Ser-2 or Ser-5 of the CTD, we found that ceTFIIE␤ induced Ser-5 phosphorylation very little but induced Ser-2 phosphorylation normally, in contrast to wild-type hTFIIE, which induced phosphorylation at both Ser-2 and Ser-5. In transcription transition assays using a linear template, ceTFIIE␤ was markedly defective in its ability to support the transition to elongation. These observations provide evidence of TFIIE involvement in the transition and suggest that Ser-5 phosphorylation is essential for Pol II to be in the processive elongation form.In eukaryotes, transcription of protein-encoding genes by RNA polymerase II (Pol II) is the first step in expression of those genes (for reviews, see references 4, 35, 44, and 51). Two sequential stages are now recognized in the establishment of Pol II processivity: transcription initiation and the transition from initiation to elongation. At initiation, five general transcription factors (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) together with Pol II form the preinitiation complex (PIC) on the core promoter. Two models of PIC formation have been proposed on the basis of recent analyses. One model involves stepwise association of the general transcription factors and Pol II on promoter DNA, while the other model entails promoter sequences binding to a preassembled Pol II holoenzyme that contains most of the general transcription factors as well as SRB (suppressor of RNA polymerase B)-and Med-containing complex (reviewed in references 3 and 22). In vitro analyses of stepwise assembly of the PIC using purified factors have demonstrated that TFIIE joins the complex at a position near the transcription start site (between positions Ϫ14 and Ϫ2), after Pol II and TFIIF have joined the complex (25, 49). TFIIE then recruits TFIIH, and these two factors stabilize and activate the PIC, resulting in isomerization of double-stranded (ds) promoter DNA (promoter me...

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“…In addition, serum-stimulated CTD kinase activity is resistant to inhibiton by the drug 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) (Dubois et al, 1994;Bonnet et al, 1999), which inhibits CDK7, CDK8, and CDK9 but not ERK-1/2 CTD kinase (Marshall and Price, 1995;Yankulov et al, 1995;Rickert et al, 1999). Finally, recombinant ERK-1/2 has been shown to ef®ciently phosphorylate the RNAP II CTD in vitro (Trigon et al, 1998;Bonnet et al, 1999). Interestingly, ERK-1/2-mediated CTD phosphorylation results in the generation of a novel RNAP IIm form that can be discriminated from the RNAP IIA and IIO forms by SDS±PAGE analysis .…”

Section: Cdks Associated With the Rnap II Transcription Machinerymentioning

confidence: 99%

“…The physiological role of RNAP IIm remains to be investigated. However, RNAP IIm appears not to be engaged in transcription and is exclusively phosphorylated at position Ser-5 (Trigon et al, 1998;Bonnet et al, 1999), whereas the pool of (transcriptionally active) RNAP IIO in unstimulated mammalian cells is phosphorylated on both Ser-5 and Ser-2 (Bregman et al, 1995;Patturajan et al, 1998;Castan Ä o et al, 2000). Finally, it is pertinent to note that CTD phophorylation by ERK-1/2 kinases has also been implicated in the repression of RNAP II in mature Xenopus oocytes as well as in rabbit and mouse zygotes prior to zygotic gene activation (Bellier et al, 1997a,b).…”

Section: Cdks Associated With the Rnap II Transcription Machinerymentioning

confidence: 99%

“…However, preferential phosphorylation of heptapeptides with a lysine residue at position 7 over consensus heptapeptide sequences has been observed with synthetic CTD peptide substrates containing only four repeats (Rickert et al, 1999). In addition, while CDK7 CTD kinase activity is generally thought to be speci®c for Ser-5 (Sun et al, 1998;Trigon et al, 1998;Ramanathan et al, 2001), there are indications that Ser-2 phosphorylation by CDK7 might occur within less conserved heptapeptide repeats located at the C-terminal end of the CTD (Dubois et al, 1997;Bensaude et al, 1999). Results of kinetic studies with P-TEFb and puri®ed RNAP II as a substrate indicated slow initial phosphorylation events followed by more rapid phosphorylation of the CTD, suggesting that prior phosphorylation events can increase P-TEFb activity (Marshall et al, 1996).…”

Section: Distinct Ctd Kinase Speci®cities and Functionsmentioning

confidence: 99%

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Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

Oelgeschläger

2002

Journal Cellular Physiology

128

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The carboxyl-terminal domain (CTD) of the largest subunit of mammalian RNA polymerase II (RNAP II) consists of 52 repeats of a consensus heptapeptide and is subject to phosphorylation and dephosphorylation events during each round of transcription. RNAP II activity is regulated during the cell cycle and cell cycledependend changes in RNAP II activity correlate well with CTD phosphorylation. In addition, global changes in the CTD phosphorylation status are observed in response to mitogenic or cytostatic signals such as growth factors, mitogens and DNA-damaging agents. Several CTD kinases are members of the cyclindependent kinase (CDK) superfamily and associate with transcription initiation complexes. Other CTD kinases implicated in cell cycle regulation include the mitogen-activated protein kinases ERK-1/2 and the c-Abl tyrosine kinase. These observations suggest that reversible RNAP II CTD phosphorylation may play a key role in linking cell cycle regulatory events to coordinated changes in transcription.

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Characterization of the Residues Phosphorylated in Vitro by Different C-terminal Domain Kinases

Cited by 108 publications

References 67 publications

cdk-7 is required for mRNA transcription and cell cycle progression in Caenorhabditis elegans embryos

cdk-7 is required for mRNA transcription and cell cycle progression in Caenorhabditis elegans embryos

Studies of Nematode TFIIE Function Reveal a Link between Ser-5 Phosphorylation of RNA Polymerase II and the Transition from Transcription Initiation to Elongation

Regulation of RNA polymerase II activity by CTD phosphorylation and cell cycle control

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