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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)

Phosphorylation of the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II has been implicated as an important step in transcriptional regulation. Previously, we reported that a cellular CTD kinase, TAK, is targeted by the human immunodeficiency virus transactivator Tat. In the present study, we analyzed several other transactivators for the ability to interact with CTD kinases in vitro. The adenovirus E1A and herpes simplex virus VP16 proteins, but not other transactivators tested, were found to associate with a cellular kinase activity that hyperphosphorylates the CTD. The interaction is dependent upon a functional activation domain of E1A or VP16, suggesting that the interaction with a CTD kinase is relevant for the transactivation function of these proteins. The CTD kinase activities that interact with E1A and VP16 are related to each other but distinct from TAK. The Tat-, E1A- and VP16-associated CTD kinase activities detected in our assay also appear unrelated to MO15, the catalytic component of the CTD kinase activity of the general transcription factor TFIIH. Thus, this study has identified a novel interaction between viral transactivators and a cellular CTD kinase and suggests that at least two CTD kinases may mediate responses to viral transactivators.
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PMID:Viral transactivators specifically target distinct cellular protein kinases that phosphorylate the RNA polymerase II C-terminal domain. 860 64

The yeast RNA polymerase III (pol III) general transcription factor TFIIIB is composed of three subunits; the TATA-binding protein (TBP)1, the TFIIB-related factor (BRF1), and a third factor termed TFIIIB90 or B". Here we report the purification of yeast TFIIIB90, cloning of the gene encoding TFIIIB90, and reconstitution of TFIIIB from recombinant polypeptides. The TFIIIB90 open reading frame encodes a 68-kDa polypeptide and has no obvious similarity to any other known protein sequences. The gene encoding TFIIIB90 is essential for viability of yeast. Using recombinant TFIIIB subunits, we found that TFIIIB90 interacts weakly with TBP in the absence of BRF1, and that this interaction is enhanced at least 25-fold by BRF1. In addition, TFIIIB90 showed pol III specificity as it could not interact with the pol II-specific TFIIB-TBP-DNA complex. To localize the regions of the TBP-DNA complex that interact with BRF1 and TFIIIB90, we tested whether the pol II factors TFIIA and TFIIB interfered with the binding of BRF1 and TFIIIB90 to TBP-DNA. Our results suggest that the binding sites for BRF1 and TFIIIB90 on TBP-DNA both overlap the binding sites for TFIIA and TFIIB.
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PMID:Cloning and functional characterization of the gene encoding the TFIIIB90 subunit of RNA polymerase III transcription factor TFIIIB. 866 56

A yeast protein has been identified that stimulates basal transcription by RNA polymerase II, binds both single- and double-stranded DNA, and interacts with both a general transcription factor and a transcriptional activator. Phosphorylation appears to regulate these interactions. The gene for the transcriptional stimulatory protein, termed TSP1, was cloned and found to be dispensable for yeast cell viability. The deduced amino acid sequence is similar to that of mammalian coactivator protein PC4.
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PMID:A yeast transcriptional stimulatory protein similar to human PC4. 870 84

Cyclin-dependent kinase (CDK) 7 was originally implicated in cell cycle control by virtue of its ability to phosphorylate and activate other CDKs. Subsequently, both CDK7 and its partner, cyclin H, were found to be associated with the general transcription factor TFIIH, suggesting additional roles for CDK7 in transcription and DNA repair. During the past year, a third subunit associated with CDK7 and cyclin H has been characterized, and the functional link between CDK7 and RNA polymerase II has been strengthened.
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PMID:Cyclin-dependent kinase 7: at the cross-roads of transcription, DNA repair and cell cycle control? 874 80

RNA polymerase (RNAP) II is subject to extensive phosphorylation on the heptapeptide repeats of the C-terminal domain (CTD) of the largest subunit. An activity that is required for the dephosphorylation of yeast RNAP II in vitro has been purified from a yeast whole cell extract by >30,000-fold. The yeast CTD phosphatase activity copurified with two bands with apparent molecular masses of 100 and 103 kDa. The properties of the yeast CTD phosphatase are similar to those of a previously characterized CTD phosphatase from HeLa cells. These properties include stimulation by the general transcription factor IIF (TFIIF), competitive inhibition by RNAP II, magnesium dependence, and resistance to okadaic acid. Both the HeLa and yeast CTD phosphatases are highly specific for their cognate polymerases. Neither phosphatase functions upon the polymerase molecule from the other species, even though the heptapeptide repeats of the CTDs in yeast RNAP II and mammalian RNAP II are essentially identical. The activity of the highly purified CTD phosphatase is stimulated >300-fold by a partially purified fraction of TFIIF. Recombinant TFIIF did not substitute for the TFIIF fraction, indicating that an additional factor present in the TFIIF fraction is required for CTD phosphatase activity. These results show that yeast contains a CTD phosphatase activity similar to that of mammalian cells that is likely composed of at least two components, one of which is 100 and/or 103 kDa.
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PMID:Purification and characterization of an RNA polymerase II phosphatase from yeast. 879 10

The RNA polymerase II general transcription factor TFIID is a multisubunit complex comprising TATA-box binding protein and associated factors (TAFIIs). In vitro experiments have suggested that TAFIIs are essential coactivators required for RNA polymerase II-directed transcription activation. Here, for the first time, we analyze systematically the in vivo function of a specific TAFII, yeast TAFII90 (yTAFII90). We show that functional inactivation of yTAFII90 by temperature-sensitive mutations or depletion leads to arrest at the G2/M phase of the cell cycle. Unexpectedly, in the absence of functional yTAFII90, a variety of endogenous yeast genes were all transcribed normally, including those driven by well-characterized activators. Taken together, our results indicate that yTAFII90 is not required for transcription activation in general, and reveal linkages between TAF function and cell-cycle progression.
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PMID:Yeast TAF(II)90 is required for cell-cycle progression through G2/M but not for general transcription activation. 882 95

Mitosis in higher eukaryotes is accompanied by a general inhibition of transcription. To begin to understand the mechanisms underlying this inhibition we have examined the behavior of the general transcription factor TFIID during mitosis. Immunocytochemistry and subcellular fractionation studies indicate that the majority of TFIID is displaced from the disassembling prophase nucleus to the mitotic cytoplasm around the time of nuclear envelope breakdown. However, a subpopulation of TFIID remains associated tightly with the condensed mitotic chromosomes. Metabolic labeling of mitotic cells revealed that several subunits of TFIID undergo mitosis-specific phosphorylation, but in spite of these changes, the TFIID complex remains intact. Functional analysis of purified TFIID from mitotic cells shows that phosphorylated forms are unable to direct activator-dependent transcription, but that this activity is restored upon dephosphorylation. These results demonstrate that TFIID regulation by phosphorylation is likely to have an important role in mitotic inhibition of RNA polymerase II transcription. In addition, they suggest a mechanism for regulating gene expression through the selective disruption of polymerase II promoter structures during mitosis.
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PMID:Mitotic regulation of TFIID: inhibition of activator-dependent transcription and changes in subcellular localization. 884 92

Spt3 of Saccharomyces cerevisiae is a factor required for normal transcription from particular RNA polymerase II-dependent promoters. Previous genetic and biochemical analyses have shown that Spt3 interacts with the yeast TATA-binding protein (TBP). To identify other factors that might interact with Spt3, we have screened for mutations that, in combination with an spt3 null mutation, lead to inviability. In this way, we have identified a mutation in MOT1, which encodes an ATP-dependent inhibitor of TBP binding to TATA boxes: Previous analyses suggested that Mot1 causes repression in vivo. However, our analysis of mot1 mutants shows that, similar to spt3 mutants, they have decreased levels of transcription from certain genes, suggesting that Mot1 may function as an activator in vivo. In addition, mot1 mutants have other phenotypes in common with spt3 delta mutants, including suppression of the insertion mutation his4-912 delta. Motivated by these Spt3-Mot1 genetic interactions, we tested for genetic interactions between Spt3 and the general transcription factor TFIIA. TFIIA has been shown previously to be functionally related to Mot1. We found that overexpression of TFIIA partially suppresses an spt3 delta mutation, that toa1 mutants have Spt-phenotypes, and that spt3 delta toa1 double mutants are inviable. We believe that, taken together, these data suggest that Spt3, Mot1, and TFIIA cooperate to regulate TBP-DNA interactions, perhaps at the level of TATA box selection in vivo.
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PMID:Evidence that Spt3 functionally interacts with Mot1, TFIIA, and TATA-binding protein to confer promoter-specific transcriptional control in Saccharomyces cerevisiae. 897 9

Five different monoclonal antibodies that immunoreact with RAP74, the large subunit of general transcription factor (TF) IIF, were produced and characterized. Using one of these antibodies, an affinity purification procedure was devised to isolate a human RNA polymerase II complex. This procedure is fast, simple, and reproducible and does not require extensive purification. The RNA polymerase II complex isolated using this procedure contains SRB (suppressor of RNA polymerase B) polypeptides, transcription factors IIE and IIF, limiting amounts of TFIIH, and the TATA-binding protein, but was devoid of TFIIB.
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PMID:Affinity purification of a human RNA polymerase II complex using monoclonal antibodies against transcription factor IIF. 911 Oct 63

TFIIH is a general transcription factor for RNA polymerase II that in addition is involved in DNA excision repair. TFIIH is composed of eight or nine subunits and we show that at least four of them, namely cdk7, cyclin H, MAT1, and p62 are localized in the coiled body, a distinct subnuclear structure that is transcription dependent and highly enriched in small nuclear ribonucleoproteins. Although coiled bodies do not correspond to sites of transcription, in vivo incorporation of bromo-UTP shows that they are surrounded by transcription foci. Immunofluorescence analysis using antibodies directed against the essential repair factors proliferating cell nuclear antigen and XPG did not reveal labeling of the coiled body in either untreated cells or cells irradiated with UV light, arguing that coiled bodies are probably not involved in DNA repair mechanisms. The localization of cyclin H in the coiled body was predominantly detected during the G1 and S-phases of the cell cycle, whereas in G2 coiled bodies were very small or not detected. Finally, both cyclin H and cdk7 did not colocalize with P80 coilin after disruption of the coiled body, indicating that these proteins are specifically targeted to the small nuclear ribonucleoprotein-containing domain.
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PMID:The cdk7-cyclin H-MAT1 complex associated with TFIIH is localized in coiled bodies. 924 2


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