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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human positive cofactor (PC4) acts as a general coactivator for activator-dependent transcription by RNA polymerase II. Here we show that PC4 coactivator function, in contrast to basal (activator-independent) transcription, is dependent both on TATA binding protein (TBP)-associated factors (TAFs) in TFIID and on TFIIH. Surprisingly, PC4 strongly represses transcription initiation by minimal preinitiation complexes in the absence of TAFs and TFIIH, while simultaneously promoting the formation of these complexes. Furthermore, TFIIH and TAFII250, the largest subunit of TFIID, can both phosphorylate PC4. These results provide evidence for an inactive, PC4-induced intermediate in preinitiation complex assembly and point to TFIIH and TAF requirements for its progression into a functional preinitiation complex. Thus PC4 coactivator activity is realized in a stepwise series of events reminiscent of prokaryotic activation pathways involving conversion of inactive RNA polymerase-promoter complexes to an initiation-competent state.
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PMID:A dynamic model for PC4 coactivator function in RNA polymerase II transcription. 948 61

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Mitotic repression of transcription of various cellular and viral gene promoters by RNA polymerase II can be reproduced in vitro either with extracts prepared from cells arrested at mitosis with the microtubule polymerization inhibitor nocodazole or with nuclear extracts prepared from asynchronous cells and the mitotic protein kinase cdc2/cyclin B. Purified cdc2/cyclin B kinase is also sufficient to inhibit transcription in reconstituted transcription reactions with biochemically purified and recombinant basal transcription factors and RNA polymerase II. The cyclin-dependent kinase inhibitor p21Waf1/Cip1/Sdi1 can reverse the effect of cdc2/cyclin B kinase, indicating that repression of transcription is due to protein phosphorylation. Transcription rescue and inhibition experiments with each of the basal factors and the polymerase suggest that multiple components of the transcription machinery are inactivated by cdc2/cyclin B kinase. For an activated promoter, targets of repression are TFIID and TFIIH, while for a basal promoter, TFIIH is the major target for mitotic inactivation of transcription. Protein labeling experiments indicate that the p62 and p36 subunits of TFIIH are in vitro substrates for mitotic phosphorylation. Using the carboxy-terminal domain of the large subunit of RNA polymerase II as a test substrate for phosphorylation, the TFIIH-associated kinase, cdk7/cyclin H, is inhibited concomitant with inhibition of transcription activity. Our results suggest that there exist multiple phosphorylation targets for the global shutdown of transcription at mitosis.
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PMID:Repression of TFIIH transcriptional activity and TFIIH-associated cdk7 kinase activity at mitosis. 948 63

pX, the hepatitis B virus (HBV)-encoded regulator, coactivates transcription through an unknown mechanism. pX interacts with several components of the transcription machinery, including certain activators, TFIIB, TFIIH, and the RNA polymerase II (POLII) enzyme. We show that pX localizes in the nucleus and coimmunoprecipitates with TFIIB from nuclear extracts. We used TFIIB mutants inactive in binding either POLII or TATA binding protein to study the role of TFIIB-pX interaction in transcription coactivation. pX was able to bind the former type of TFIIB mutant and not the latter. Neither of these sets of TFIIB mutants supports transcription. Remarkably, the latter TFIIB mutants fully block pX activity, suggesting the role of TFIIB in pX-mediated coactivation. By contrast, in the presence of pX, TFIIB mutants with disrupted POLII binding acquire the wild-type phenotype, both in vivo and in vitro. These results suggest that pX may establish the otherwise inefficient TFIIB mutant-POLII interaction, by acting as a molecular bridge. Collectively, our results demonstrate that TFIIB is the in vivo target of pX.
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PMID:Hepatitis B virus pX targets TFIIB in transcription coactivation. 948 73

The cell cycle is regulated by various protein kinases, including cyclin-dependent kinases (CDKs). D-type CDKs, CDK4, and CDK6, phosphorylate retinoblastoma protein and are believed to regulate through the G1 phase of the cell cycle. CDK inhibitor p16INK4A has been characterized as binding CDK4 and CDK6 and as inhibiting phosphorylation of retinoblastoma protein by these CDKs. Thus p16INK4A is implicated in regulating the cell cycle at the G1 phase. The largest subunit of RNA polymerase II (pol II) contains an essential C-terminal domain (CTD). General transcription factor TFIIH, which contains CDK7, phosphorylates the CTD in vitro. The CTD phosphorylation is shown to be involved in transcriptional regulation in vivo and in vitro. Phosphorylation of RNA pol II CTD by TFIIH is thought to play an important role in transcriptional regulation. Here we report that p16INK4A associates with RNA pol II CTD and TFIIH. p16(INK4A) inhibited the CTD phosphorylation by TFIIH. These findings suggest that p16INK4A may regulate transcription via CTD phosphorylation in the cell cycle.
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PMID:Cyclin-dependent kinase inhibitor p16INK4A inhibits phosphorylation of RNA polymerase II by general transcription factor TFIIH. 948 60

The C-terminal part of the largest subunit of eukaryotic RNA polymerase II is composed solely of the highly repeated consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. This domain, called the C-terminal domain (CTD), is phosphorylated mostly at serine residues during transcription initiation, but the precise role of this phosphorylation remains controversial. Several protein kinases are able to phosphorylate this sequence in vitro. The aim of this work was to define the positions of the amino acids phosphorylated by four of these CTD kinases (transcription factor (TF) IIH-kinase, DNA-dependent protein kinase, and the mitogen-activated protein kinases ERK1 and ERK2) and to compare the specificity of these different protein kinases. We show that TFIIH kinase and the mitogen-activated protein kinases phosphorylate only serine 5 of the CTD sequence, whereas DNA-dependent protein kinase phosphorylates serines 2 and 7. Among the different CTD kinases, only TFIIH kinase is appreciably more active on two repeats of the consensus sequence than on one motif. These in vitro results can provide some clues to the nature of the protein kinases responsible for the in vivo phosphorylation of the RNA polymerase CTD. In particular, the ratio of phosphorylated serine to threonine observed in vivo cannot be explained if TFIIH kinase is the only protein kinase involved in the phosphorylation of the CTD.
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PMID:Characterization of the residues phosphorylated in vitro by different C-terminal domain kinases. 950 78

The transactivator protein Tat stimulates transcriptional elongation from the HIV-1 LTR. One mechanism by which Tat increases HIV-1 transcription is by interacting with RNA polymerase II and TFIIH to increase phosphorylation of the polymerase C-terminal domain. Recent studies indicate that specific elongation factors may also be required to modulate Tat function. Here, we used biochemical analysis and in vitro transcription assays to identify cellular factors required for Tat activation. This analysis resulted in the purification of a cellular factor Tat-CT1 which is a human homolog of the yeast transcription factor SPT5. Immunodepletion of Tat-CTl from HeLa extract demonstrated that this factor was involved in transcriptional activation by Tat. However, the absence of this factor from HeLa extract did not prevent transcriptional activation by VP16. These findings are consistent with a model in which Tat-mediated effects on transcriptional elongation are mediated in part by the action of the human homolog of the yeast transcription factor SPT5.
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PMID:Role of the human homolog of the yeast transcription factor SPT5 in HIV-1 Tat-activation. 951 52

Saccharomyces cerevisiae Gal11, a component of the holoenzyme of RNA polymerase II, interacts through its functional domains A and B with the small (Tfa2) and large (Tfa1) subunits of the general transcription factor (TF) IIE, respectively. We have recently suggested that Gal11 functions through a common pathway with TFIIE in transcriptional regulation (Sakurai, H., and Fukasawa, T. (1997) J. Biol. Chem. 272, 32663-32669). Here, we report that the activity of the TFIIH-associated kinase, responsible for phosphorylation of the largest subunit of RNA polymerase II at the carboxyl-terminal domain (CTD), is enhanced cooperatively by Gal11 and TFIIE. The enhancement of CTD phosphorylation was observed in the holoenzyme of RNA polymerase II, but not in its core enzyme. The stimulatory effect was completely abolished in the absence of either domain B of Gal11 or the Tfa1 subunit of TFIIE, suggesting that the domain B-Tfa1 interaction is necessary, if not sufficient, for an extensive phosphorylation of the CTD by TFIIH. Stimulation of basal transcription by Gal11 was coupled with enhancement of TFIIH-catalyzed CTD phosphorylation in a cell-free transcription system, suggesting that Gal11 activates transcription by stimulating the CTD phosphorylation in the cell.
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PMID:Functional correlation among Gal11, transcription factor (TF) IIE, and TFIIH in Saccharomyces cerevisiae. Gal11 and TFIIE cooperatively enhance TFIIH-mediated phosphorylation of RNA polymerase II carboxyl-terminal domain sequences. 954 82

The HIV-1 Tat protein is an RNA-binding transcriptional transactivator. Recent findings suggest that Tat associates with a cellular kinase that phosphorylates the carboxyl-terminal domain of the largest subunit of RNA polymerase II. Here we review, in brief, the role of Tat-associated kinase in Tat-activated transcription. We discuss evidence that suggests involvement of TFIIH and/or P-TEFb.
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PMID:Tat, Tat-associated kinase, and transcription. 957 May 10

P-TEFb is required for the transition from abortive elongation into productive elongation and is capable of phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II. We cloned a cDNA encoding the large subunit of Drosophila P-TEFb and found the predicted protein contained a cyclin motif. We now name the large subunit cyclin T and the previously cloned small subunit (Zhu, Y. R., Peery, T., Peng, J. M., Ramanathan, Y., Marshall, N., Marshall, T., Amendt, B., Mathews, M. B., and Price, D. H. (1997) Genes Dev. 11, 2622-2632) cyclin-dependent kinase 9 (CDK9). Recombinant P-TEFb produced in baculovirus-transfected Sf9 cells exhibited 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole-sensitive kinase activity similar to native P-TEFb. Kc cell nuclear extract depleted of P-TEFb failed to generate long DRB-sensitive transcripts, but this activity was restored upon addition of either native or recombinant P-TEFb. Like other CDKs, CDK9 is essentially inactive in the absence of its cyclin partner. P-TEFb containing a CDK9 mutation that knocked out the kinase activity did not function in transcription. Deletion of the carboxyl-terminal domain of cyclin T in P-TEFb reduced both the kinase and transcription activity to about 10%. The CDK-activating kinase in TFIIH was unable to activate the CTD kinase activity of P-TEFb.
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PMID:Identification of a cyclin subunit required for the function of Drosophila P-TEFb. 959 31

Transcription initiation by RNA polymerase II (RNA pol II) requires interaction between cis-acting promoter elements and trans-acting factors. The eukaryotic promoter consists of core elements, which include the TATA box and other DNA sequences that define transcription start sites, and regulatory elements, which either enhance or repress transcription in a gene-specific manner. The core promoter is the site for assembly of the transcription preinitiation complex, which includes RNA pol II and the general transcription fctors TBP, TFIIB, TFIIE, TFIIF, and TFIIH. Regulatory elements bind gene-specific factors, which affect the rate of transcription by interacting, either directly or indirectly, with components of the general transcriptional machinery. A third class of transcription factors, termed coactivators, is not required for basal transcription in vitro but often mediates activation by a broad spectrum of activators. Accordingly, coactivators are neither gene-specific nor general transcription factors, although gene-specific coactivators have been described in metazoan systems. Transcriptional repressors include both gene-specific and general factors. Similar to coactivators, general transcriptional repressors affect the expression of a broad spectrum of genes yet do not repress all genes. General repressors either act through the core transcriptional machinery or are histone related and presumably affect chromatin function. This review focuses on the global effectors of RNA polymerase II transcription in yeast, including the general transcription factors, the coactivators, and the general repressors. Emphasis is placed on the role that yeast genetics has played in identifying these factors and their associated functions.
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PMID:Molecular genetics of the RNA polymerase II general transcriptional machinery. 961 49


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