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)

Octamer binding transcription factors (Oct factors) play important roles in activation of transcription of various genes but, in some cases, require cofactors that interact with the DNA binding (POU) domain. In the present study, a yeast two-hybrid screen with the Oct-1 POU domain as a bait identified MAT1 as a POU domain-binding protein. MAT1 is known to be required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK), which is functionally associated with the general transcription factor IIH (TFIIH). Further analyses showed that MAT1 interacts with POU domains of Oct-1, Oct-2, and Oct-3 in vitro in a DNA-independent manner. MAT1-containing TFIIH was also shown to interact with POU domains of Oct-1 and Oct-2. MAT1 is shown to enhance the ability of a recombinant CDK7-cyclin H complex (bipartite CAK) to phosphorylate isolated POU domains, intact Oct-1, and the C-terminal domain of RNA polymerase II, but not the originally defined substrate, CDK2. Phosphopeptide mapping indicates that the site (Ser385) of a mitosis-specific phosphorylation that inhibits Oct-1 binding to DNA is not phosphorylated by CAK. However, one CAK-phosphorylated phosphopeptide comigrates with a Cdc2-phosphorylated phosphopeptide previously shown to be mitosis-specific, suggesting that, in vitro, CAK is able to phosphorylate at least one site that is also phosphorylated in vivo. These results suggest (i) that interactions between POU domains and MAT1 can target CAK to Oct factors and result in their phosphorylation, (ii) that MAT1 not only functions as a CAK assembly factor but also acts to alter the spectrum of CAK substrates, and (iii) that a POU-MAT1 interaction may play a role in the recruitment of TFIIH to the preinitiation complex or in subsequent initiation and elongation reactions.
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PMID:The cyclin-dependent kinase-activating kinase (CAK) assembly factor, MAT1, targets and enhances CAK activity on the POU domains of octamer transcription factors. 936 58

The tumor suppressor protein p53 acts as a transcriptional activator that can mediate cellular responses to DNA damage by inducing apoptosis and cell cycle arrest. p53 is a nuclear phosphoprotein, and phosphorylation has been proposed to be a means by which the activity of p53 is regulated. The cyclin-dependent kinase (CDK)-activating kinase (CAK) was originally identified as a cellular kinase required for the activation of a CDK-cyclin complex, and CAK is comprised of three subunits: CDK7, cyclin H, and p36MAT1. CAK is part of the transcription factor IIH multiprotein complex, which is required for RNA polymerase II transcription and nucleotide excision repair. Because of the similarities between p53 and CAK in their involvement in the cell cycle, transcription, and repair, we investigated whether p53 could act as a substrate for phosphorylation by CAK. While CDK7-cyclin H is sufficient for phosphorylation of CDK2, we show that p36MAT1 is required for efficient phosphorylation of p53 by CDK7-cyclin H, suggesting that p36MAT1 can act as a substrate specificity-determining factor for CDK7-cyclin H. We have mapped a major site of phosphorylation by CAK to Ser-33 of p53 and have demonstrated as well that p53 is phosphorylated at this site in vivo. Both wild-type and tumor-derived mutant p53 proteins are efficiently phosphorylated by CAK. Furthermore, we show that p36 and p53 can interact both in vitro and in vivo. These studies reveal a potential mechanism for coupling the regulation of p53 with DNA repair and the basal transcriptional machinery.
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PMID:p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner. 937 54

The Srb10-Srb11 protein kinase of Saccharomyces cerevisiae is a cyclin-dependent kinase (cdk)-cyclin pair which has been found associated with the carboxy-terminal domain (CTD) of RNA polymerase II holoenzyme forms. Previous genetic findings implicated the Srb10-Srb11 kinase in transcriptional repression. Here we use synthetic promoters and LexA fusion proteins to test the requirement for Srb10-Srb11 in repression by Ssn6-Tup1, a global corepressor. We show that srb10delta and srb11delta mutations reduce repression by DNA-bound LexA-Ssn6 and LexA-Tup1. A point mutation in a conserved subdomain of the kinase similarly reduced repression, indicating that the catalytic activity is required. These findings establish a functional link between Ssn6-Tup1 and the Srb10-Srb11 kinase in vivo. We also explored the relationship between Srb10-Srb11 and CTD kinase I (CTDK-I), another member of the cdk-cyclin family that has been implicated in CTD phosphorylation. We show that mutation of CTK1, encoding the cdk subunit, causes defects in transcriptional repression by LexA-Tup1 and in transcriptional activation. Analysis of the mutant phenotypes and the genetic interactions of srb10delta and ctk1A suggests that the two kinases have related but distinct roles in transcriptional control. These genetic findings, together with previous biochemical evidence, suggest that one mechanism of repression by Ssn6-Tup1 involves functional interaction with RNA polymerase II holoenzyme.
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PMID:Functional relationships of Srb10-Srb11 kinase, carboxy-terminal domain kinase CTDK-I, and transcriptional corepressor Ssn6-Tup1. 948 31

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 transition from abortive into productive elongation is proposed to be controlled by a positive transcription elongation factor b (P-TEFb) through phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Drosophila P-TEFb was identified recently as a cyclin-dependent kinase (CDK9) paired with a cyclin subunit (cyclin T). We demonstrate here the cloning of multiple cyclin subunits of human P-TEFb (T1 and T2). Cyclin T2 has two forms (T2a and T2b) because of alternative splicing. Both cyclin T1 and T2 are ubiquitously expressed. Immunoprecipitation and immunodepletion experiments carried out on HeLa nuclear extract (HNE) indicated that cyclin T1 and T2 were associated with CDK9 in a mutually exclusive manner and that almost all CDK9 was associated with either cyclin T1 or T2. Recombinant CDK9/cyclin T1, CDK9/cyclin T2a, and CDK9/cyclin T2b produced in Sf9 cells possessed DRB-sensitive kinase activity and functioned in transcription elongation in vitro. Either cyclin T1 or T2 was required to activate CDK9, and the truncation of the carboxyl terminus of the cyclin reduced, but did not eliminate, P-TEFb activity. Cotransfection experiments indicated that all three CDK9/cyclin combinations dramatically activated the CMV promoter.
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PMID:Identification of multiple cyclin subunits of human P-TEFb. 949 9

The activation of cyclin-dependent kinases (CDKs) requires phosphorylation of a threonine residue within the T-loop catalyzed by CDK-activating kinases (CAKs). Thus far no functional CAK homologue has been reported in plants. We screened an Arabidopsis cDNA expression library for complementation of a budding yeast CAK mutant. A cDNA, cak1At, was isolated that suppressed the CAK mutation in budding yeast, and it also complemented a fission yeast CAK mutant. cak1At encodes a protein related to animal CAKs. The CAK similarity was restricted to the conserved kinase domains, leading to classification of Cak1At as a distinct CDK in the phylogenetic tree. Immunoprecipitates with the anti-Cak1At antibody phosphorylated human CDK2 at the threonine residue (T160) within the T-loop and activated its activity to phosphorylate histone H1. Whereas CAKs in animals and fission yeast are involved in regulation of the cell cycle and basal transcription by phosphorylating the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II, Cak1At did not phosphorylate the CTD. An Arabidopsis CTD-kinase isolated separately from Cak1At was shown to interact with the yeast protein p13(suc1), but it had no CDK2-kinase activity. Therefore, the CTD of RNA polymerase II is probably phosphorylated by a Cdc2-related kinase distinct from Cak1At. cak1At is a single-copy gene in Arabidopsis and is highly expressed in proliferating cells of suspension cultures.
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PMID:A distinct cyclin-dependent kinase-activating kinase of Arabidopsis thaliana. 956 Feb 21

TAK, a multisubunit cellular protein kinase that specifically associates with the human immunodeficiency virus Tat proteins and hyperphosphorylates the carboxyl-terminal domain of RNA polymerase II, is a cofactor for Tat and mediates its transactivation function. The catalytic subunit of TAK has been identified as cyclin-dependent kinase Cdk9, and its regulatory partner has been identified as cyclin T1; these proteins are also components of positive transcription elongation factor P-TEFb. TAK activity is up-regulated upon activation of peripheral blood lymphocytes and following macrophage differentiation of promonocytic cell lines. We have found that activation of peripheral blood lymphocytes results in increased mRNA and protein levels of both Cdk9 and cyclin T1. Cdk9 and cyclin T1 induction occurred in purified CD4(+) primary T cells activated by a variety of stimuli. In contrast, phorbol ester-induced differentiation of promonocytic cell lines into macrophage-like cells produced a large induction of cyclin T1 protein expression from nearly undetectable levels, while Cdk9 protein levels remained at a constant high level. Measurements of cyclin T1 mRNA levels in a promonocytic cell line suggested that regulation of cyclin T1 occurs at a posttranscriptional level. These results suggest that cyclin T1 and TAK function may be required in differentiated monocytes and further show that TAK activity can be regulated by distinct mechanisms in different cell types.
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PMID:Tat-associated kinase, TAK, activity is regulated by distinct mechanisms in peripheral blood lymphocytes and promonocytic cell lines. 981 24

TFIIH is a multisubunit complex, containing ATPase, helicases, and kinase activities. Functionally, TFIIH has been implicated in transcription by RNA polymerase II (RNAPII) and in nucleotide excision repair. A member of the cyclin-dependent kinase family, CDK7, is the kinase subunit of TFIIH. Genetically, CDK7 homologues have been implicated in transcription in Saccharomyces cerevisiae, and in mitotic regulation in Schizosaccharomyces pombe. Here we show that in mitosis the CDK7 subunit of TFIIH and the largest subunit of RNAPII become hyperphosphorylated. MPF-induced phosphorylation of CDK7 results in inhibition of the TFIIH-associated kinase and transcription activities. Negative and positive regulation of TFIIH requires phosphorylation within the T-loop of CDK7. Our data establishes TFIIH and its subunit CDK7 as a direct link between the regulation of transcription and the cell cycle.
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PMID:The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7. 983 6

The growth suppressor p53 is an important key element which controls cell cycle progression in response to cellular stress like DNA damage. Its ability to act as transcriptional activator or repressor links transcription and cell cycle control. Several target genes selectively transactivated by p53 are implicated in growth control, apoptosis and DNA repair. Here we report the interaction of p53 with another important dual player of cell cycle control and transcription, the protein kinase complex CDK7/cyclin H/Mat1 (CDK activating kinase, CAK kinase). This is implicated in the activating phosphorylation of CDK2/cyclin A kinase required to allow cells to proceed through the G1/S transition, and on the other hand, as a component of the basal transcription factor TFIIH found to be necessary for CTD phosphorylation of RNA polymerase II in order to allow elongation of transcription. Based on previous binding studies of p53 with other C-terminal interaction partners of p53 we demonstrate a direct physical interaction of p53 with cyclin H in vitro and in vivo. As a consequence of this interaction we tested the influence of p53 on the kinase activity of CAK kinase for CTD and CDK2 phosphorylation. The addition of wild type p53 to the kinase reactions resulted in a significant downregulation of CDK2 phosphorylation and CTD phosphorylation by the CDK activating kinase. On the other hand addition of a mutant p53His175 failed to downregulate CDK2 and CTD phosphorylation by the CDK activating kinase. In an attempt to support our findings in vivo we measured CAK kinase activity in p21-/- and p53-/- mice embryonal fibroblasts under conditions when p53 gets activated by irradiation. In the case of p21-/- cells this led to a significant reduction of CTD phosphorylation activity of the CDK activating kinase by irradiation of the cells. On the other hand in p53 cells no downregulation of CTD phosphorylation activity of CAK kinase was observed indicating that this kind of negative regulation of CAK kinase activity is exclusively due to a functional p53. These findings imply a direct involvement of p53 in triggering growth arrest by its interaction with the CDK activating kinase complex without the need of cyclin-dependent kinase inhibitors (CKIs) and potentially suggest a new mechanism for p53-dependent apoptosis.
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PMID:Regulation of CAK kinase activity by p53. 984 Sep 37

The rates of transcription of several protein coding genes during Acanthamoeba differentiation have been examined by nuclear run-on and RNase protection assays. During early encystment, transcription by RNA polymerase II increases approximately 4-fold, whereas transcription by RNA polymerases I and III is decreased, as previously described. The rates of transcription from a wide variety of individual genes are only slightly affected during the first 16 h of encystment, although profilin gene expression is markedly increased. The levels of mRNAs encoding TPBF, TATA binding protein, cyclin-dependent kinase, protein disulfide isomerase, profilin, myosin II heavy chain, ubiquitin and extendin are stable during mature cyst formation, whereas mRNAs encoding actin, S-adenosyl methionine synthase and tubulin are substantially decreased in abundance within 16 h of starvation-induced encystment. We conclude that in contrast to the negative regulation of large rRNA and 5S rRNA synthesis during differentiation, the RNA polymerase II transcription apparatus is not negatively regulated. Control of Acanthamoeba differentiation is likely to be mediated by positive regulation of genes necessary for cyst maturation.
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PMID:Transcription by RNA polymerase II during Acanthamoeba differentiation. 987 98


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