Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Yeast RNA polymerase II initiation factor b copurifies with three polypeptides of 85, 73, and 50 kilodaltons and with a protein kinase that phosphorylates the carboxyl-terminal repeat domain (CTD) of the largest polymerase subunit. The gene that encodes the 73-kilodalton polypeptide, designated TFB1, was cloned and found to be essential for cell growth. The deduced protein sequence exhibits no similarity to those of protein kinases. However, the sequence is similar to that of the 62-kilodalton subunit of the HeLa transcription factor BFT2, suggesting that this factor is the human counterpart of yeast factor b. Immunoprecipitation experiments using antibodies to the TFB1 gene product demonstrate that the transcriptional and CTD kinase activities of factor b are closely associated with an oligomer of the three polypeptides. Photoaffinity labeling with 3'-O-(4-benzoyl)benzoyl-ATP (adenosine triphosphate) identified an ATP-binding site in the 85-kilodalton polypeptide, suggesting that the 85-kilodalton subunit contains the catalytic domain of the kinase.
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PMID:Cloning of a subunit of yeast RNA polymerase II transcription factor b and CTD kinase. 144

The largest subunit of eukaryotic RNA polymerase II contains a carboxyl-terminal domain (CTD) which is comprised of repetitive heptapeptides with a consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We demonstrate here that the mouse CTD expressed in and purified from Escherichia coli can be phosphorylated in vitro by a p34cdc2/CDC28-containing CTD kinase from mouse ascites tumor cells. The product of this reaction, a phosphorylated form of the CTD, contains phosphoserine and phosphothreonine, but not phosphotyrosine. The same phosphoamino acid content is observed in the in vivo phosphorylated CTD from a mouse cell line. Synthetic peptides with naturally occurring non-consensus heptapeptide sequences can also be phosphorylated by CTD kinase in vitro. Phosphoamino acid analysis of these non-consensus heptapeptides together with direct sequencing of a phosphorylated heptapeptide reveals that serines (or threonines) at positions two and five are the sites phosphorylated by mouse CTD kinase. Thus, the -Ser(Thr)-Pro- motif common to p34cdc2/CDC28-containing protein kinases is the recognition site for mouse CTD kinase.
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PMID:Identification of phosphorylation sites in the repetitive carboxyl-terminal domain of the mouse RNA polymerase II largest subunit. 189 39

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II can be phosphorylated by a p34cdc2/CDC28-containing CTD kinase. Phosphorylated serine (or threonine) is located at positions 2 and 5 in the repetitive heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show here that phosphorylation of the mouse CTD retards its electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels in a way similar to that observed for the II0 form of the largest subunit of RNA polymerase II phosphorylated in vivo. At the maximum level of phosphorylation by CTD kinase in vitro, there are 15-20 phosphates evenly distributed among the 52 heptapeptide repeats that comprise the mouse CTD. Gel filtration chromatography and sucrose gradient ultracentrifugation analyses indicate that phosphorylation induces a dramatic conformational change in the CTD with the phosphorylated form adopting a far more extended structure than the unphosphorylated CTD.
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PMID:Phosphorylation causes a conformational change in the carboxyl-terminal domain of the mouse RNA polymerase II largest subunit. 198 83

Changes in protein kinase activities are thought to contribute to the alteration of gene expression after heat shock and related stresses. In an attempt to identify enzymes which might be involved in both chromatin structure modification and transcriptional switch in heat-shocked cells, we have studied protein kinase activities in heat-shocked cell lysates with two exogenous substrates: a tetramer of a heptapeptide (heptapeptide 4) corresponding to the RNA polymerase II C-terminal domain (CTD), and the histone H1. Heat-shock and arsenite stress were found to stimulate strongly CTD kinase activity. H1 kinase activity was also stimulated but more weakly. Stimulation of CTD and H1 kinases occurs mainly at the early phase of recovery and by a process which is independent of protein synthesis. The stress-induced H1 kinase is shown to contain a molecule related to the mitotic-promoting factor (MPF) Cdc2 component. On the other hand, though Cdc2-related protein has also been reported to be part of a CTD kinase complex, we show that the stress-induced CTD kinase activity corresponds to a distinct entity. It is proposed that stress activation of CTD kinase might be involved in changing the specificity of RNA polymerase II.
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PMID:Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts. 217 28

The unique C-terminal repeat domain (CTD) of the largest subunit (IIa) of eukaryotic RNA polymerase II consists of multiple repeats of the heptapeptide consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats ranges from 26 in yeast to 42 in Drosophila to 52 in mouse. The CTD is essential in vivo, but its structure and function are not yet understood. The CTD can be phosphorylated at multiple serine and threonine residues, generating a form of the largest subunit (II0) with markedly reduced mobility in NaDodSO4/polyacrylamide gels. To investigate this extensive phosphorylation, which presumably modulates functional properties of RNA polymerase II, we began efforts to purify a specific CTD kinase. Using CTD-containing fusion proteins as substrates, we have purified a CTD kinase from the yeast Saccharomyces cerevisiae. The enzyme extensively phosphorylates the CTD portion of both the fusion proteins and intact subunit IIa, producing products with reduced electrophoretic mobilities. The properties of the CTD kinase suggest that it is distinct from previously described protein kinases. Analogous activities were also detected in Drosophila and HeLa cell extracts.
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PMID:A protein kinase that phosphorylates the C-terminal repeat domain of the largest subunit of RNA polymerase II. 265 24

The Src-homology (SH) 2 domain, found in a variety of proteins, has a binding site for phosphotyrosine-containing peptides. In adaptor proteins such as Grb2, the SH2 domain plays an important role in the assembly of signal transducer complexes. Many nonreceptor tyrosine kinases--e.g., Abl and Src--also contain SH2 domains. Without a functional SH2 domain, these tyrosine kinases retain catalytic activity but lose their biological function. This result suggests that the SH2 domain may be involved in the selection of biologically relevant substrates. We have previously shown that the carboxyl-terminal repeated domain (CTD) of the mammalian RNA polymerase II is a substrate for the Abl but not the Src tyrosine kinase. This specificity is conferred in part by the SH2 domain. The Abl SH2 domain binds the tyrosine-phosphorylated [Tyr(P)] CTD and is required for the processive and stoichiometric phosphorylation of the 52 tyrosines in the CTD. Mutation of the Abl SH2 or exchanging it with that of Src, which does not bind the Tyr(P)-CTD, abolished processivity and reduced the CTD kinase activity without any effect on autophosphorylation or the phosphorylation of nonspecific substrates. These results demonstrate that the SH2 domain of the Abl tyrosine kinase plays an active role in catalysis and suggests that SH2 domain and the tyrosine kinase domain may act in concert to confer substrate specificity.
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PMID:Src homology 2 domain as a specificity determinant in the c-Abl-mediated tyrosine phosphorylation of the RNA polymerase II carboxyl-terminal repeated domain. 753 94

An array of tandem heptapeptide repeats at the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II constitute a highly conserved structure essential for viability. Studies have established that the CTD is phosphorylated at different stages of the transcription cycle, and that it may be involved in transcriptional regulation. The exact role of the CTD remains elusive, as in vitro reconstituted transcription using the adenovirus major late promoter does not require the CTD. Previous studies showed that transcription from the murine dihydrofolate reductase (DHFR) promoter can be only accomplished by the form of RNA polymerase II that contains the hypophosphorylated CTD (RNAPIIA), but not by the form that lacks it (RNAPIIB). Here we show that the CTD, but not its phosphorylation, is required for initiation of transcription. We also show that transcription requires CTD kinase activity provided by the CDK subunit of TFIIH.
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PMID:Requirement for TFIIH kinase activity in transcription by RNA polymerase II. 756 58

RNA polymerase (RNAP) II is a multisubunit enzyme composed of several different subunits. Phosphorylation of the C-terminal domain (CTD) of the largest subunit is tightly regulated. In quiescent or in exponentially growing cells, both the unphosphorylated (IIa) and the multiphosphorylated (IIo) subunits of RNAP II are found in equivalent amounts as the result of the equilibrated antagonist action of protein kinases and phosphatases. In Drosophila and mammalian cells, heat shock markedly modifies the phosphorylation of the RNAP II CTD. Mild heat shocks result in dephosphorylation of the RNAP II CTD. This dephosphorylation is blocked in the presence of actinomycin D, as the CTD dephosphorylation observed in the presence of protein kinase inhibitors. Thus, heat shock might inactivate CTD kinases which are operative at normal growth temperatures, as some protein kinase inhibitors do. In contrast, severe heat shocks are found to increase the amount of phosphorylated subunit independently of the transcriptional activity of the cells. Mild and severe heat shocks activate protein kinases, which then phosphorylate, in vitro and in vivo, the CTD fused to beta-galactosidase. Most of the heat-shock-activated CTD kinases present in cytosolic lysates co-purify with the activated mitogen-activated protein (MAP) kinases, p42mapk and p44mapk. The weak CTD kinase activation occurring upon mild heat shock might be insufficient to compensate for the heat inactivation of the already existing CTD kinases. However, under severe stress, the MAP kinases are strongly heat activated and might prevail over the phosphatases. A survey of different cells and different heat-shock conditions shows that the RNAP II CTD hyperphosphorylation rates follow the extent of MAP kinase activation. These observations lead to the proposal that the RNAP II CTD might be an in vivo target for the activated p42mapk and p44mapk MAP kinases.
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PMID:Phosphorylation state of the RNA polymerase II C-terminal domain (CTD) in heat-shocked cells. Possible involvement of the stress-activated mitogen-activated protein (MAP) kinases. 758 77

The Saccharomyces cerevisiae gene KIN28 is a member of the cyclin-dependent kinase (CDK) family. The Kin28 protein shares extensive sequence identity with the vertebrate CDK-activating kinase MO15 (Cdk7), which phosphorylates CDKs in vitro on a critical threonine residue. Kin28 and MO15 have recently been found to copurify with the transcription factor IIH (TFIIH) holoenzyme of yeast and human cells, respectively. Although TFIIH is capable of phosphorylating the C-terminal domain (CTD) of RNA polymerase II, it has been unclear whether Kin28 is the physiologically relevant CTD kinase or what role CTD phosphorylation plays in transcription. In this study, we used a thermosensitive allele of KIN28 and a hemagglutinin epitope-tagged Kin28 protein to investigate Kin28 function in transcription and in the cell cycle. We show that Kin28 acts as a positive regulator of mRNA transcription in vivo and possesses CTD kinase activity in vitro. However, Kin28 neither regulates the phosphorylation state of the yeast cell cycle CDK, Cdc28, nor possesses CDK-activating kinase activity in vitro. We conclude that Kin28 is a strong candidate for the physiological CTD kinase of S. cerevisiae and that Kin28 function is required for mRNA transcription.
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PMID:KIN28 encodes a C-terminal domain kinase that controls mRNA transcription in Saccharomyces cerevisiae but lacks cyclin-dependent kinase-activating kinase (CAK) activity. 776 Jul 96

Phosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II has been suggested to be critical for transcription initiation, activation, or elongation. A kinase activity specific for CTD is a component of the general transcription factor TFIIH. Recently, a cyclin-dependent kinase-activator kinase (MO15 and cyclin H) was found to be associated with TFIIH preparations and was suggested to be the CTD kinase. TFIIH preparations containing mutant, kinase-deficient MO15 lack CTD kinase activity, indicating that MO15 is critical for polymerase phosphorylation. Nonetheless, these mutant TFIIH preparations were fully functional (in vitro) in both basal and activated transcription. These results indicate that CTD phosphorylation is not required for transcription with a highly purified system.
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PMID:A kinase-deficient transcription factor TFIIH is functional in basal and activated transcription. 776 69


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