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)

The predicted amino acid sequence of the vaccinia virus gene A18R shows significant homology to the human ERCC3 gene product, which is a member of the DEXH subfamily of the DNA and RNA helicase superfamily II and which plays a role in both RNA polymerase II transcription and nucleotide excision repair of DNA. The vaccinia virus A18R gene product is expressed throughout infection and is encapsidated in virions. Vaccinia virions containing mutant A18R gene product are defective in early viral transcription in vitro, and infection with A18R mutant virus results in aberrant viral transcription late during infection. Thus we hypothesize that the vaccinia virus A18R gene product is a helicase that plays a role in viral transcription and possibly DNA repair. As a first test of this hypothesis, we have affinity purified an amino-terminal polyhistidine-tagged A18R protein and shown that it has DNA-dependent ATPase activity. The A18R ATPase activity is stimulated by both single-stranded and double-stranded DNA and by RNA.DNA hybrids, but not by either single-stranded or double-stranded RNA.
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PMID:The vaccinia virus A18R gene product is a DNA-dependent ATPase. 782 83

The C31 subunit belongs to a complex of three subunits (C31, C34 and C82) specific to RNA polymerase (pol) III that have no counterparts in other RNA polymerases. This complex is thought to play a role in transcription initiation since it interacts with the general initiation factor TFIIIB via subunit C34. We have obtained a conditional mutation of pol III by partially deleting the acidic C-terminus of the C31 subunit. A Saccharomyces cerevisiae strain carrying this truncated C31 subunit is impaired in in vivo transcription of tRNAs and failed to grow at 37 degrees C. This conditional growth phenotype was suppressed by overexpression of the gene coding for the largest subunit of pol III (C160), suggesting an interaction between C160 and C31. The mutant pol III enzyme transcribed non-specific templates at wild-type rates in vitro, but was impaired in its capacity to transcribe tRNA genes in the presence of general initiation factors. Transcription initiation, but not termination or recycling of the enzyme, was affected in the mutant, suggesting that it could be altered on interaction with initiation factors or on the formation of the open complex. Interestingly, the C-terminal deletion was also suppressed by a high gene dosage of the DED1 gene encoding a putative helicase.
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PMID:A mutation in the C31 subunit of Saccharomyces cerevisiae RNA polymerase III affects transcription initiation. 783 45

The role of nucleotides in activated RNA polymerase II transcription was studied. Permanganate footprinting confirmed that there is a strict nucleotide requirement for forming open promoter complexes that cannot be overcome by the addition of a dinucleotide primer corresponding to the start site sequence. However, higher concentrations of other nucleoside triphosphates can substitute for ATP in catalyzing open complex formation. Opening catalyzed by these nucleotides is inhibited by the ATP analogue adenosine 5'-O-(thio-triphosphate), suggesting that they may function through cross-binding to the ATP site. The KM for ATP for opening and the involvement of other nucleotides in opening differs from the characteristics reported for TFIIH helicase and C-terminal domain kinase activities. This raises the possibility that opening does not involve these activities. The results alleviate very significantly the considerable current uncertainty concerning the role of ATP in the mammalian mRNA transcription initiation pathway.
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PMID:Nucleotide requirements for activated RNA polymerase II open complex formation in vitro. 783 91

The first open reading frame of the blueberry scorch carlavirus (BBScV) genome encodes a putative replication-associated protein of 223 kDa (p223). A pulse-chase analysis of viral RNA translated in vitro in rabbit reticulocyte lysate revealed that p223 was proteolytically processed. Using a full-length ORF 1 cDNA clone in a coupled in vitro transcription/translation reaction, we confirmed that the ORF 1 gene product of BBScV processes autocatalytically. From sequence alignments with phylogenetically related viruses, including tymoviruses, we predicted that p223 contained a papain-like proteinase domain with a putative catalytic cysteine994 and histidine1075. A second possible proteinase domain, which contained cysteine895 and histidine984 residues with similar spacing but was otherwise less similar to the viral papain-like proteinases, was identified immediately upstream of the predicted catalytic site. The cleavage site of the proteinase was predicted to be between the putative helicase and the polymerase domains, possibly between or close to glycine1472 and alanine1473. Supporting these predictions, deletion of the 2091 nucleotides encoding the C-terminal region of p223, which contained the putative RNA polymerase domain and the putative cleavage site of the polyprotein, abolished autoproteolysis. Deletion of the 2061 nucleotides encoding the N-terminal region, which contained the putative methyltransferase domain, did not affect autoproteolysis. Alteration of cysteine994, histidine1075, or glycine1472 abolished autoproteolysis in vitro, supporting the involvement of these residues at the catalytic site and cleavage site. Alteration of the upstream cysteine895 and histidine984 residues did not affect processing in vitro. Capped BBScV full-length transcripts containing mutations in the codons for either cysteine994 or histidine1075 were not infectious in the systemic host plants Chenopodium quinoa and C. amaranticolor, whereas alteration of glycine1472 signficantly decreased but did not abolish infectivity. Transcripts containing mutations in the codons for either cysteine895 or histidine984 also were infectious, but resulted in delayed symptom expression in plants.
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PMID:Autocatalytic processing of the 223-kDa protein of blueberry scorch carlavirus by a papain-like proteinase. 787 21

The 130-kDa mfd gene product is required for coupling transcription to repair in Escherichia coli. Mfd displaces E. coli RNA polymerase (Pol) stalled at a lesion, binds to the damage recognition protein UvrA, and increases the template strand repair rate during transcription. Here, the interactions of Mfd (transcription-repair coupling factor, TRCF) with DNA, RNA Pol, and UvrA were investigated. TRCF bound nonspecifically to double stranded DNA; binding to DNA produced alternating DNase I-protected and -hypersensitive regions, suggesting possible wrapping of the DNA around the enzyme. Weaker binding to single stranded DNA and no binding to single stranded RNA were observed. DNA binding required ATP, and hydrolysis of ATP promoted dissociation. Removal of a stalled RNA Pol also requires ATP hydrolysis. Apparently, TRCF recognizes a stalled elongation complex by directly interacting with RNA Pol, since binding to a synthetic transcription bubble was no stronger than binding to double stranded DNA, and binding to free RNA Pol holoenzyme and to initiation and elongation complexes in the absence of adenosine 5'-O-(thiotriphosphate) were observed. Structure-function analysis showed that residues 379-571 are involved in binding to a stalled RNAP. The helicase motifs region, residues 571-931, binds to ATP and duplex polynucleotide (DNA:DNA or DNA:RNA). Dissociation of the ternary complex upon hydrolysis of ATP also requires the carboxyl terminus of TRCF. Finally, residues 1-378 bind to UvrA and deliver the damage recognition component of the excision nuclease to the lesion.
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PMID:Structure and function of transcription-repair coupling factor. I. Structural domains and binding properties. 787 61

The RNA polymerase II large subunit contains an essential carboxy-terminal domain (CTD) believed to be involved in the response to regulators during transcription initiation. The CTD is phosphorylated on a portion of RNA polymerase II molecules in vivo and it can be phosphorylated by the general transcription factor TFIIH in vitro. A highly purified TFIIH from rat liver has been described; this, like human and yeast TFIIH, contains associated CTD kinase and helicase activities. We report here that two polypeptides of the purified mammalian TFIIH are the MO15/Cdk7 kinase and cyclin H subunits of the Cdk-activating kinase Cak, previously identified as a positive regulator of Cdc2 and Cdk2. TFIIH and Cak preparations are each capable of phosphorylating recombinant CTD and recombinant Cdk2 proteins. The presence of Cak in TFIIH indicates that Cak may have roles in transcriptional regulation and in cell-cycle control.
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PMID:Association of Cdk-activating kinase subunits with transcription factor TFIIH. 788 50

A basal repressor of class II gene transcription was identified, purified, and found to be identical to nonhistone chromosomal protein HMG2. HMG2 was shown to inhibit basal transcription under conditions in which transcription templates form soluble complexes with HMG2. Order-of-addition experiments clearly revealed that HMG2 acted after assembly of a TBP-TFIIA-promoter complex and before formation of the fourth phosphodiester bond by RNA polymerase II. Subsequently, an activity that efficiently counteracted repression of transcription by HMG2 in both TBP- and TFIID-containing transcription systems was isolated. Several lines of evidence suggested that antirepression was mediated by a TFIIH-associated factor. The antirepressor first coeluted with TFIIH, was depleted from this fraction by antibodies directed against the TFIIH subunit p62, was dependent on either ATP or dATP, and then was inhibited by the ATP analogs AMP-PNP and ATP gamma S. Relief of HMG2-mediated repression as well as basal promoter function of TFIIH may involve a helicase that coelutes with TFIIH and displays similar nucleotide specificities. Taken together, these data suggest novel consequences of chromatin-associated HMG proteins and they provide direct evidence for a role of TFIIH-associated enzymes in ATP-dependent antirepression of nonhistone chromosomal proteins.
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PMID:Repression of basal transcription by HMG2 is counteracted by TFIIH-associated factors in an ATP-dependent process. 800 73

Escherichia coli Rho factor is required for termination of transcription at certain sites by RNA polymerase. Binding to unstructured cytosine-containing RNA target sites, subsequent RNA-dependent ATP hydrolysis, and an RNA-DNA helicase activity that presumably facilitates termination, are considered essential for Rho function. Yet the RNA recognition elements have remained elusive, the parameters relating RNA binding to ATPase activation have been obscure, and the mechanistic steps that integrate Rho's characteristics with its termination function in vitro and in vivo have been largely undefined. Recent work offers new insights into these interactions with results that are both surprising and satisfying in the context of Rho's emerging structure. These include the requirements for binding and ATPase activation by a variety of RNA substrates, dynamic analyses of Rho tracking, helicase and termination activity, and the participation of a new factor (NusG) that interacts with Rho. Models for Rho function are considered in the light of these recent revelations.
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PMID:Rho and RNA: models for recognition and response. 802 88

The RNA polymerase II general transcription factor TFIIH is composed of several polypeptides. The observation that the largest subunit of TFIIH is the excision-repair protein XPB/ERCC3 (ref. 1), a helicase implicated in the human DNA-repair disorders xeroderma pigmentosum (XP) and Cockayne's syndrome, suggests a functional link between transcription and DNA repair. To understand the connection between these two cellular processes, we have extensively purified and functionally analysed TFIIH. We find that TFIIH has a dual role, being required for basal transcription of class II genes and for participation in DNA-excision repair. TFIIH is shown to complement three different cell extracts deficient in excision repair: XPB/ERCC3, XPC and XPD/ERCC2. The complementation of XPB and XPD is a consequence of ERCC3 and ERCC2 being integral subunits of TFIIH, whereas complementation of XPC is due to an association of this polypeptide with TFIIH. We found that the general transcription factor IIE negatively modulates the helicase activity of TFIIH through a direct interaction between TFIIE and the ERCC3 subunit of TFIIH.
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PMID:Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II. 815 90

Using a defined RNA polymerase II (pol II) transcription system, we have investigated the roles of basal factors at discrete stages during the transcription cycle (e.g., initiation, promoter clearance, and transcript elongation). Abortive initiation assays revealed that TATA-binding protein, transcription factors TFIIB and TFIIF, and pol II were necessary and sufficient to form functional initiation complexes on both linear and supercoiled templates. By contrast, TFIIE, TFIIH, and ATP hydrolysis were additionally required during promoter clearance from linear templates, while negative supercoiling obviated the need for these auxiliary factors. Furthermore, TFIIE, TFIIH, and supercoiling were not required during elongation. Our results suggest a role for TFIIH-associated helicase activity or supercoiling during promoter clearance rather than open complex formation. These results establish abortive initiation as a useful assay for studying functional initiation complex formation in defined eukaryotic transcription systems and provide a framework for investigating regulation at different stages of the eukaryotic transcription cycle.
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PMID:Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II. 815 90


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