A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription.

Abstract

(13)(14)(15) and were suggested to be responsible for the kinase activity of TFIIH. Furthermore, a Saccharomyces cerevisiae M015-related kinase, KIN28, was identified as a p33 kinase subunit of yeast TFIIH (16). The TFIIH-associated KIN28 is included in the holo-TFIIH associated with transcription but can be separated from a form of TFIIH still functional in DNA repair (17).The C-terminal domain (CTD) of Pol II is a substrate for the TFIIH kinase (18), and one of the functions of TFIIH may be to regulate polymerase activity by modulating CTD phosphorylation. The exact role of CTD phosphorylation in transcription remains elusive. The CTD of polymerase entering the preinitiation complex (19) or stalled close to the promoter (20) is not phosphorylated, whereas the elongating polymerase is highly phosphorylated (21). In addition, CTD that is not phosphorylated interacts with the TATA-binding protein (TBP) component of TFIID (22) and with TFIIE (23), leading to the notion that phosphorylation of CTD would be critical for promoter clearance. On the other hand, kinase inhibitor studies have suggested that CTD phosphorylation is not required for basal transcription (24). Another model suggested by studies with yeast polymerase holoenzymes suggests thatThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.CTD phosphorylation might be involved in activated transcription (25,26). Here the role of M015 and cyclin H in CTD phosphorylation and the effects of this phosphorylation on transcription are discussed. MATERIALS AND METHODSAntibodies. A monoclonal antibody (mAb) against p62 (mAb 3c9) was provided by J.-M. Egly (Centre National de la Recherche Scientifique, Illkirch, France); the M015 polyclonal rabbit serum was raised against a 15-amino-acid Cterminal peptide; the cyclin H polyclonal rabbit serum was raised against a glutathione S-transferase (GST)-cyclin H fusion protein; the p89 antiserum was a polyclonal rabbit serum against a GST-p89-N-terminal fragment; and commercial preparations of the polyclonal rabbit CDK2 antibody (M2; Santa Cruz, Biotechnology, Santa Cruz, CA) and Myc mAb 9E10 (Cambridge Research Biochemicals) were used.Transfections. Fifteen micrograms of plasmids encoding wild-type or mutant MO15 cDNAs with C-terminal triple Myc-epitope tags (46) in pcDNA3 (Invitrogen) or 15 ,ug of pcDNA3 together with 15 ,g of an untagged cyclin H cDNA in pcDNA3 were transfected into human bone sarcoma U-2 OS cells by calcium phosphate coprecipitation. This particular cell line was used because of its high transfectability. Fortyeight hours after transfection, cells were washed in phosphatebuffered saline and lysed in ELB (150 mM NaCl/50 mM Hepes, pH 7.5/5 mM EDTA/0.1% Nonidet P-40 containing 10 Ag each of aprotinin and leupeptice per ml, 0.2 mM phenylmethylsulfonyl fluoride, 10 mM f3-glycerophosphate, and 5 mM NaF) for immunoprecipitation.Immunoblotting and Immunopre...

The Rate of c-fos Transcription in Vivo Is Continuously Regulated at the Level of Elongation by Dynamic Stimulus-coupled Recruitment of Positive Transcription Elongation Factor b

“…Initiation is defined as the recruitment of pol II to the promoter and the synthesis of the first RNA phosphodiester bonds, which are molecular mechanisms that include CTD Ser-5 phosphorylation (31,32). To further characterize the initiation steps necessary for c-fos transcription, we investigated the assembly of the PIC by looking for the association of specific GTFs with the 5Ј region of the gene (Fig.…”

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

“…CDK7 homologues in other organisms activate transcription by phosphorylating the CTD of RNAPII (3,(5)(6)(7). To test whether C. elegans cdk-7 functions similarly, we examined the phosphorylation state of RNAPII in cdk-7(ax224) mutant embryos.…”

“…Subsequently, CDK7 was also found to be a component of the general transcription factor TFIIH (3)(4)(5)(6). As part of TFIIH, CDK7 phosphorylates the C-terminal domain (CTD) of RNA polymerase II (RNAPII) (3,(5)(6)(7). The in vitro CDK-and CTD-kinase activities of CDK7 appear to be widely conserved, and have been demonstrated in systems ranging from Schizosaccharomyas pombe (8,9) to rice (10) and mammals (11).…”

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

“…In vitro, CTD phosphorylation by TFIIH appears to generally stimulate RNAP II transcription (Orphanides et al, 1996;Coin and Egly, 1998;Riedl and Egly, 2000), but has also been reported to inhibit RNAP II activity under certain conditions (Serizawa et al, 1993;Gebara et al, 1997). Moreover, although association with the trimeric CAK complex is required for TFIIH transcription activity in vitro (Svejstrup et al, 1995;Drapkin et al, 1996;Reardon et al, 1996), TFIIH CTD kinase activity is dispensable for transcription of some genes both in vitro (Akoulitchev et al, 1995;Ma È kela È et al, 1995) and in yeast cells (Lee and Lis, 1998). This observations is consistent with the well documented fact that the CTD requirement for RNAP II transcription in vitro varies between different promoters (Thompson et al, 1989;Buermeyer et al, 1992;Kang and Dahmus, 1993;Akoulitchev et al, 1995).…”