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)

Nuclear transcription is repressed when eukaryotic cells enter mitosis. Using Xenopus egg extracts shifted to the mitotic state with recombinant cyclin B1 protein, we have been able to reproduce mitotic repression of transcription in vitro. Active RNA polymerase III transcription is observed in interphase extracts in the absence of added cyclin, but is strongly repressed by the induction of cdc2/cyclin B (maturation/mitosis promoting factor, MPF) kinase activity in the mitotic extract. Studies with protein kinase inhibitors show that protein phosphorylation is required for repression. Add-back experiments indicate that repression of class III gene transcription is due to inactivation of the transcription factor TFIIIB. TFIIIB is composed of the TATA-box binding protein (TBP) and TBP-associated factors of 75 and 92 kDa. In the present study, we show that TBP and a polypeptide of 92 kDa are substrates of the mitotic kinase in highly purified TF- IIIB fractions. We also show that a phosphatase present in the Xenopus egg extract can reactivate transcription after repression by the mitotic kinases. This result suggests a mechanism for reactivation of transcription after exit from mitosis into the G1 phase of the cell cycle. As for pol III genes, purified cdc2/cyclin B kinase is sufficient to inhibit transcription by RNA polymerase II in a reconstituted transcription system containing the basal transcription factors and polymerase.
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PMID:Repression of RNA polymerase II and III transcription during M phase of the cell cycle. 898 11

Cdc2 kinase triggers the entry of mammalian cells into mitosis, the only cell cycle phase in which transcription is globally repressed. We show here that Cdc2 kinase phosphorylates components of the RNA polymerase II transcription machinery including the RNA polymerase II carboxy-terminal repeat domain (CTD). To test specifically the effect of CTD phosphorylation by Cdc2 kinase, we used a yeast in vitro transcription extract that is dependent on exogenous RNA polymerase II that contains a CTD. Phosphorylation was carried out using immobilized Cdc2 so that the kinase could be removed from the phosphorylated polymerase. ATP gamma S and Cdc2 kinase were used to produce an RNA polymerase IIO that was not detectably dephosphorylated in the transcription extract. RNA polymerase IIO produced in this way was defective in promoter-dependent transcription, suggesting that phosphorylation of the CTD by Cdc2 kinase can mediate transcription repression during mitosis. In addition, we show that phosphorylation of pol II with the human TFIIH-associated kinase Cdk7 also decreases transcription activity despite a different pattern of CTD phosphorylation by this kinase. These results extend previous findings that RNA polymerase IIO is defective in preinitiation complex formation. Here we demonstrate that phosphorylation of the CTD by cyclin-dependent kinases with different phosphoryl acceptor specificities can inhibit transcription in a CTD-dependent transcription system.
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PMID:Phosphorylation of the carboxy-terminal repeat domain in RNA polymerase II by cyclin-dependent kinases is sufficient to inhibit transcription. 905 97

The Schizosaccharomyces pombe gene pch1(+) (pombe cyclin C homology) was isolated in a two-hybrid screen for proteins that interact with Cdc2. The cyclin box region of Pch1 protein shares greatest sequence identity with mammalian and Drosophila C-type cyclins ( approximately 33% identity). Pch1 is significantly less similar to Mcs2 (19% identity), a second member of the C-type cyclin family in S. pombe. Cdc2 co-precipitates with Pch1 in S. pombe cell lysates, although Cdc2 may not be the major catalytic partner of a Pch1 kinase in vivo. Purified Pch1-associated kinase phosphorylated myelin basic protein, histone H1, and a peptide corresponding to the carboxyl-terminal domain repeat of RNA polymerase II. The amount of pch1 mRNA does not oscillate during the cell cycle, as is the case for mRNA transcripts of other C-type cyclin genes. Deltapch1 cells are inviable, therefore S. pombe has two essential genes that encode members of the C-type cyclin family, pch1(+) and mcs2(+). The Deltapch1 mutation causes pleiotropic morphological defects and an associated growth deficiency, but loss of Pch1 activity does not result in a cdc cell cycle-arrest phenotype.
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PMID:Pch1(+), a second essential C-type cyclin gene in Schizosaccharomyces pombe. 911 79

The molecular mechanisms that regulate the cardiomyocyte cell cycle and its terminal differentiation remain largely unknown. To determine which cyclins or cyclin dependent kinases (CDKs) are important for cardiomyocyte proliferation, we examined the expression of cyclins and CDKs during normal cardiac development. All cyclins and CDKs were highly expressed during embryonic cardiac development, then they decreased at different rates after birth. The mRNAs and proteins of cyclins A and B (G2 and M phase cyclins) were found in embryonic and neonatal hearts, but were not detected in young or adult hearts. In contrast, while the mRNAs of cyclins D1, D2, D3, and E (G1 and S phase cyclins) were observed during all stages of development, the proteins of cyclins D1, D3, and E were observed in hearts at the young growth stage, although the levels decreased differently. Reverse transcriptase-polymerase chain reaction (RT-PCR) using specific cyclin B and D3 primers revealed that cyclins B and D3 originated from cardiomyocytes and noncardiomyocytes. The CDKs (cdc2, CDK2, and CDK4) were highly expressed during embryonic cardiac development and maintained almost constant levels during neonatal periods. However, they were expressed at very low levels at the young and adult stages. The pattern of proliferating cell nuclear antigen (PCNA) expression during cardiac development was similar to the expression of CDKs. These findings suggest that all cyclins and CDKs are involved in the cardiac cell cycle, and that marked and rapid reduction of mitotic cyclins may be associated with the withdrawal of the cardiac cell cycle after birth.
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PMID:Cyclins and cyclin dependent kinases during cardiac development. 926 23

We have previously identified a cellular protein kinase activity termed TAK that specifically associates with the HIV types 1 and 2 Tat proteins. TAK hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II in vitro in a manner believed to activate transcription [Herrmann, C. H. & Rice, A. P. (1995) J. Virol. 69, 1612-1620]. We show here that the catalytic subunit of TAK is a known human kinase previously named PITALRE, which is a member of the cyclin-dependent family of proteins. We also show that TAK activity is elevated upon activation of peripheral blood mononuclear cells and peripheral blood lymphocytes and upon differentiation of U1 and U937 promonocytic cell lines to macrophages. Therefore, in HIV-infected individuals TAK may be induced in T cells following activation and in macrophages following differentiation, thus contributing to high levels of viral transcription and the escape from latency of transcriptionally silent proviruses.
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PMID:TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines. 935 49

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

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

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

Previous studies have shown that the apoptotic response of cells following DNA damage requires p53 expression. Wild-type p53 protein levels increase in response to DNA damage and its growth-suppressive action is thought to be mediated by transcriptional activation of the p21/WAF1/CIP1 gene, the product of which is a potent inhibitor of cyclin-dependent kinases. The mechanism by which elevated p53 levels lead to apoptosis is not known, but is believed to involve transcriptional activation of apoptotic genes, such as BAX. We have studied transformed human cells that constitutively express high levels of the R273H mutant p53, which has been reported to lack transcriptional activation activity. We used the inability to induce the p21/Waf1/Cip1 protein as a marker to verify the lack of transcriptional activation activity. Cells expressing the R273H mutant of p53 do not show an increase in p21/Waf1/Cip1 following irradiation with ionizing or UVB radiation. Surprisingly, these cells are very susceptible to induction of apoptosis by UVB radiation, as seen by the formation of a nucleosomal ladder and the proteolytic cleavage of poly(ADP-ribose) polymerase. This suggests that the R273 mutant p53 can function normally in apoptosis but not in transcriptional activation following DNA damage. Furthermore, an inhibitor of RNA polymerase II is a potent inducer of apoptosis in these cells, demonstrating that transcription is not required for apoptosis and suggesting that stalled RNA polymerase II complexes can initiate apoptosis. Interestingly, proteolytic cleavage of p53 occurs during apoptosis in these cells, generating a 45-kDa fragment and liberating the DNA repair helicase binding domain of p53. We propose that the peptide liberated from the carboxy terminus of p53 may contribute to its apoptotic activity, possibly through interaction with the XPB and XPD DNA helicases.
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PMID:The apoptotic and transcriptional transactivation activities of p53 can be dissociated. 949 57


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