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)

Cyclin-dependent kinase (CDK)-activating kinases (CAKs) carry out essential activating phosphorylations of CDKs such as Cdc2 and Cdk2. The catalytic subunit of mammalian CAK, MO15/Cdk7, also functions as a subunit of the general transcription factor TFIIH. However, these functions are split in budding yeast, where Kin28p functions as the kinase subunit of TFIIH and Cak1p functions as a CAK. We show that Kin28p, which is itself a CDK, also contains a site of activating phosphorylation on Thr-162. The kinase activity of a T162A mutant of Kin28p is reduced by approximately 75 to 80% compared to that of wild-type Kin28p. Moreover, cells containing kin28(T162A) and a conditional allele of TFB3 (the ortholog of the mammalian MAT1 protein, an assembly factor for MO15 and cyclin H) are severely compromised and display a significant further reduction in Kin28p activity. This finding provides in vivo support for the previous biochemical observation that MO15-cyclin H complexes can be activated either by activating phosphorylation of MO15 or by binding to MAT1. Finally, we show that Kin28p is no longer phosphorylated on Thr-162 following inactivation of Cak1p in vivo, that Cak1p can phosphorylate Kin28p on Thr-162 in vitro, and that this phosphorylation stimulates the CTD kinase activity of Kin28p. Thus, Kin28p joins Cdc28p, the major cell cycle Cdk in budding yeast, as a physiological Cak1p substrate. These findings indicate that although MO15 and Cak1p constitute different forms of CAK, both control the cell cycle and the phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II by TFIIH.
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PMID:Activating phosphorylation of the Kin28p subunit of yeast TFIIH by Cak1p. 1037 27

TFIIH is an RNA polymerase II transcription factor that performs ATP-dependent functions in both transcription initiation, where it catalyzes formation of the open complex, and in promoter escape, where it suppresses arrest of the early elongation complex at promoter-proximal sites. TFIIH possesses three known ATP-dependent activities: a 3' --> 5' DNA helicase catalyzed by its XPB subunit, a 5' --> 3' DNA helicase catalyzed by its XPD subunit, and a carboxyl-terminal domain (CTD) kinase activity catalyzed by its CDK7 subunit. In this report, we exploit TFIIH mutants to investigate the contributions of TFIIH DNA helicase and CTD kinase activities to efficient promoter escape by RNA polymerase II in a minimal transcription system reconstituted with purified polymerase and general initiation factors. Our findings argue that the TFIIH XPB DNA helicase is primarily responsible for preventing premature arrest of early elongation intermediates during exit of polymerase from the promoter.
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PMID:A role for the TFIIH XPB DNA helicase in promoter escape by RNA polymerase II. 1042 72

The general transcription factor (TF) IIE is required for mRNA synthesis of many, but not all, genes in yeast. In the transcription process, TFIIE regulates TFIIH kinase activity that phosphorylates the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. The CTD and the CTD kinase Kin28, a subunit of TFIIH, have been shown to be dispensable for activation of several heat shock genes and the copper metallothionein gene CUP1. Here we analyzed requirement of TFIIE for transcription of these genes and found that TFIIE is necessary for activation of the heat shock genes by heat shock transcription factor Hsf1. By contrast, transcription of CUP1 mediated by both Hsf1 and copper-activated transcription factor Ace1 was inducible after inactivating TFIIE. These results show that both TFIIE and the CTD/the CTD kinase exhibit "gene specificities" which are overlapping, but not identical to each other, and thereby suggest that TFIIE functions with or without involvement of the CTD/the CTD kinase depending on the gene to be transcribed.
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PMID:Activator-specific requirement for the general transcription factor IIE in yeast. 1044 94

A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein. Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation. Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD. Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts. We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain. These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism. This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.
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PMID:Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation. 1053 63

HIV-1 gene expression relies upon a complex machinery that is primarily controlled by two viral regulatory proteins, Tat and Rev. Rev is involved in regulating post-transcriptional events of HIV-1 gene expression. The Tat protein transactivates transcription from the HIV-1 5' long terminal repeat (LTR) and acts in synergy with specific cellular factors. Recently, it has been shown that one set of these cellular factors is a protein kinase activity termed TAK (Tat-associated kinase), which activates transcription by hyperphosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II. TAK also enhances transcription of HIV-2, together with the retroviral transactivator, Tat-2. The TAK activity appears to be related to the CTD kinase P-TEFb, which stabilizes transcription elongation of many genes and was originally isolated from Drosophila extracts. Both TAK and P-TEFb contain at least two subunits: the cyclin-dependent kinase, CDK9 (PITALRE), the catalytic subunit, and the regulatory subunit, cyclin T1. CDK9 and cyclin T1 are ubiquitous factors that affects many cellular processes, including cell differentiation and apoptosis. The involvement of TAK in HIV-1 and HIV-2 gene expression is an important aspect in the biology of these two retroviruses, and may lead to the development of novel antiretroviral drugs and/or gene therapy approaches for the treatment of patients with AIDS.
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PMID:Regulatory functions of Cdk9 and of cyclin T1 in HIV tat transactivation pathway gene expression. 1053 59

The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important. We sought to identify the CTD kinase responsible for capping enzyme targeting. The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro. However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not. Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo. Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable. Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.
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PMID:Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II. 1059 13

Recent evidence suggests a role for the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II) in pre-mRNA processing. The yeast NRD1 gene encodes an essential RNA-binding protein that shares homology with mammalian CTD-binding proteins and is thought to regulate mRNA abundance by binding to a specific cis-acting element. The present work demonstrates genetic and physical interactions among Nrd1p, the pol II CTD, Nab3p, and the CTD kinase CTDK-I. Previous studies have shown that Nrd1p associates with the CTD of pol II in yeast two-hybrid assays via its CTD-interaction domain (CID). We show that nrd1 temperature-sensitive alleles are synthetically lethal with truncation of the CTD to 9 or 10 repeats. Nab3p, a yeast hnRNP, is a high-copy suppressor of some nrd1 temperature-sensitive alleles, interacts with Nrd1p in a yeast two-hybrid assay, and coimmunoprecipitates with Nrd1p. Temperature-sensitive alleles of NAB3 are suppressed by deletion of CTK1, a kinase that has been shown to phosphorylate the CTD and increase elongation efficiency in vitro. This set of genetic and physical interactions suggests a role for yeast RNA-binding proteins in transcriptional regulation.
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PMID:A yeast heterogeneous nuclear ribonucleoprotein complex associated with RNA polymerase II. 1065 11

Alkalization of the medium is associated with and required for the cellular development to meiosis and sporulation in the yeast Saccharomyces cerevisiae. To elucidate the molecular mechanisms for the significance of external alkalization, we isolated mutants defective in division arrest at G1 phase under an alkaline condition. The mutants obtained had recessive alleles of SRB10 encoding the cyclin (SRB11)-dependent protein kinase that phosphorylates the CTD domain of the largest subunit of RNA polymerase II and negatively regulates the transcriptional initiation of certain genes. A delta srb11 deletion mutant showed the same cell cycle defect. When shifted to alkali, wild-type cells decreased transcript levels of G1-cyclin genes (CLN1 to CLN3) and KIN28-CCL1 (encoding another CTD kinase-cyclin pair which, in contrast, stimulates the promoter clearance and transcriptional elongation in most genes), resulting in the accumulation of G1 cells and the hypophosphorylated form of RNA polymerase II and in an increase in cell size. However, under the same conditions, a delta srb10 mutant was defective in these events, except the downregulation of CLN1 and CLN2. The delta srb10 mutation also influenced on the transcript levels of meiosis-inducing genes called IME1 and IME2: the mutation elevated the transcript level of IME1 but reduced that of IME2, resulting in partial defects in premeiotic DNA synthesis and meiosis. Overexpression of KIN28 and CCL1 in wild-type cells impaired the alkali-induced G1 arrest and the rate of meiosis and elevated the transcript levels of SRB11 and IME1. These results indicate that a transcriptional autoregulatory loop for KIN28-CCL1 and SRB10-SRB11 is important for G1 arrest and meiosis. We also found that environmental conditions for meiosis finely regulate the transcript levels of KIN28 and CCL1, such that nitrogen starvation first elevates them but subsequent alkalization of medium decreases them.
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PMID:A transcriptional autoregulatory loop for KIN28-CCL1 and SRB10-SRB11, each encoding RNA polymerase II CTD kinase-cyclin pair, stimulates the meiotic development of S. cerevisiae. 1086 6

We report that the chromatin-specific transcription elongation factor FACT functions in conjunction with the RNA polymerase II CTD kinase P-TEFb to alleviate transcription inhibition by DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor). We find that the kinase activity of TFIIH is dispensable for this activity, demonstrating that TFIIH-mediated CTD phosphorylation is not involved in the regulation of FACT and DSIF/NELF activities. Thus, we propose a novel transcriptional regulatory network in which DSIF/NELF inhibition of transcription is prevented by P-TEFb in cooperation with FACT. This study uncovers a novel role for FACT in the regulation of transcription on naked DNA that is independent of its activities on chromatin templates. In addition, this study reveals functional differences between P-TEFb and TFIIH in the regulation of transcription.
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PMID:FACT relieves DSIF/NELF-mediated inhibition of transcriptional elongation and reveals functional differences between P-TEFb and TFIIH. 1091 1

Cyclin-dependent kinase 7 (Cdk7) forms a trimeric complex with cyclin H and Mat1 to form the mammalian Cdk-activating kinase, CAK, as well as a part of the basal transcription factor TFIIH, where Cdk7 phosphorylates the C-terminal domain (CTD) of the large subunit of RNA polymerase II. Here, we report a novel interaction between Cdk7 and a histidine triad (HIT) family protein, Hint/PKCI-1. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation and subcellular localization studies, where overexpression of Cdk7 leads to partial relocalization of Hint to the nucleus. The physical association is independent of cyclin H binding or Cdk7 kinase activity and is conserved between the related Sacharomyces cerevisiae CTD kinase Kin28 and the HIT protein Hnt1. Furthermore, combination of a disruption of HNT1 and a KIN28 temperature-sensitive allele in S. cerevisiae led to highly elongated cell morphology and reduced colony formation, indicating a genetic interaction between KIN28 and HNT1. The physical and genetic interactions of Hint and Hnt1 with Cdk7 and Kin28 suggest a role for this class of histidine triad proteins in the regulation of Cdk7 and Kin28 functions.
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PMID:Interactions of Cdk7 and Kin28 with Hint/PKCI-1 and Hnt1 histidine triad proteins. 1095 87


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