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)

We recently identified a novel rat cDNA: rab1B, closely related to the rab1A cDNA and to the yeast YPT1 gene. The rab1B cDNA encodes a 202 amino acid protein (22.1 kDa) that was produced in Escherichia coli under the control of the phi 10 promoter for the T7 RNA polymerase. The rab1B protein was purified in large amounts to near homogeneity in a simplified procedure. We studied the biochemical properties of rab1B and rab1A proteins. They both bind specifically GTP and GDP and possess intrinsic GTPase activities. The rab1B Lys21----Met mutant protein does not bind GTP, whereas the Ala65----Thr mutant has a reduced GTPase activity and is competent for autophosphorylation in the presence of GTP.
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PMID:Biochemical properties of the YPT-related rab1B protein. Comparison with rab1A. 250 43

A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase IIA has been partially purified and characterized. The kinase has a native molecular weight of about 200 kilodaltons. This kinase utilizes Mg2+ and ATP and transfers about 20 phosphates to the heptapeptide repeats Pro-Thr-Ser-Pro-Ser-Tyr-Ser in the carboxyl-terminal domain of the 220-kilodalton subunit of soybean RNA polymerase II. This phosphorylation results in a mobility shift of the 220-kilodalton subunits of a variety of eukaryotic RNA polymerases to polypeptides ranging in size from greater than 220 kilodaltons to 240 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels. The phosphorylation is highly specific to the heptapeptide repeats since a degraded subunit polypeptide of 180 kilodaltons that lacks the heptapeptide repeats is poorly phosphorylated. Synthetic heptapeptide repeat multimers inhibit the phosphorylation of the 220-kilodalton subunit.
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PMID:A protein kinase from wheat germ that phosphorylates the largest subunit of RNA polymerase II. 253 25

Two genomic sequences that share homology with Rp11215, the gene encoding the largest subunit of RNA polymerase II in Drosophila melanogaster, have been isolated from the nematode Caenorhabditis elegans. One of these sequences was physically mapped on chromosome IV within a region deleted by the deficiency mDf4, 25 kilobases (kb) from the left deficiency breakpoint. This position corresponds to ama-1 (resistance to alpha-amanitin), a gene shown previously to encode a subunit of RNA polymerase II. Northern (RNA) blotting and DNA sequencing revealed that ama-1 spans 10 kb, is punctuated by 11 introns, and encodes a 5.9-kb mRNA. A cDNA clone was isolated and partially sequenced to confirm the 3' end and several splice junctions. Analysis of the inferred 1,859-residue ama-1 product showed considerable identity with the largest subunit of RNAP II from other organisms, including the presence of a zinc finger motif near the amino terminus, and a carboxyl-terminal domain of 42 tandemly reiterated heptamers with the consensus Tyr Ser Pro Thr Ser Pro Ser. The latter domain was found to be encoded by four exons. In addition, the sequence oriented ama-1 transcription with respect to the genetic map. The second C. elegans sequence detected with the Drosophila probe, named rpc-1, was found to encode a 4.8-kb transcript and hybridized strongly to the gene encoding the largest subunit of RNA polymerase III from yeast, implicating rpc-1 as encoding the analogous peptide in the nematode. By contrast with ama-1, rpc-1 was not deleted by mDf4 or larger deficiencies examined, indicating that these genes are no closer than 150 kb. Genes flanking ama-1, including two collagen genes, also have been identified.
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PMID:Molecular cloning and sequencing of ama-1, the gene encoding the largest subunit of Caenorhabditis elegans RNA polymerase II. 258 13

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 C-terminal domain of mammalian RNA polymerase subunit IIa consists of 52-tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This C-terminal domain is essentially unmodified in RNA polymerase IIA and extensively phosphorylated in RNA polymerase IIO. A monoclonal antibody directed against the C-terminal domain was shown by kinetic enzyme-linked immunosorbent assay to have a 10-fold higher reactivity with RNA polymerase IIA than with RNA polymerase IIO. The ability of increasing concentrations of this monoclonal antibody to inhibit the initiation and elongation phase of transcription was determined. Although both phases of the transcription reaction were inhibited, a 10-fold higher concentration of antibody was required to inhibit elongation than was required to inhibit initiation. These results support the hypothesis that RNA polymerase IIA, containing an unphosphorylated C-terminal domain, is involved in the formation of an initiated complex, whereas elongation is catalyzed by RNA polymerase IIO, containing a phosphorylated C-terminal domain. Further indication that the C-terminal domain undergoes a structural change during the transcription cycle results from the observation that this domain is 3-fold more sensitive to clostripain cleavage in the elongation enzyme than in the free enzyme.
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PMID:Transcription-dependent structural changes in the C-terminal domain of mammalian RNA polymerase subunit IIa/o. 270 35

Four complementation groups of temperature-sensitive (ts) mutants of Sindbis virus that fail to make RNA at the nonpermissive temperature are known, and we have previously shown that group F mutants have defects in nsP4. Here we map representatives of groups A, B, and G. Restriction fragments from a full-length clone of Sindbis virus, Toto1101, were replaced with the corresponding fragments from the various mutants. These hybrid plasmids were transcribed in vitro by SP6 RNA polymerase to produce infectious RNA transcripts, and the virus recovered was tested for temperature sensitivity. After each lesion was mapped to a specific region, cDNA clones of both mutants and revertants were sequenced in order to determine the precise nucleotide change responsible for each mutation. Synthesis of viral RNA and complementation by rescued mutants were also examined in order to study the phenotype of each mutation in a uniform genetic background. The single mutant of group B, ts11, had a defect in nsP1 (Ala-348 to Thr). All of the group A and group G mutants examined had lesions in nsP2 (Ala-517 to Thr in ts17, Cys-304 to Tyr in ts21, and Gly-736 to Ser in ts24 for three group A mutants, and Phe-509 to Leu in ts18 and Asp-522 to Asn in ts7 for two group G mutants). In addition, ts7 had a change in nsP3 (Phe-312 to Ser) which also rendered the virus temperature sensitive and RNA-. Thus, changes in any of the four nonstructural proteins can lead to failure to synthesize RNA at a nonpermissive temperature, indicating that all four are involved in RNA synthesis. From the results presented here and from previous results, several of the activities of the nonstructural proteins can be deduced. It appears that nsP1 may be involved in the initiation of minus-strand RNA synthesis. nsP2 appears to be involved in the initiation of 26S RNA synthesis, and in addition it appears to be a protease that cleaves the nonstructural polyprotein precursors. It may also be involved in shutoff of minus-strand RNA synthesis. nsP4 appears to function as the viral polymerase or elongation factor. The functions of nsP3 are as yet unresolved.
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PMID:Mapping of RNA- temperature-sensitive mutants of Sindbis virus: assignment of complementation groups A, B, and G to nonstructural proteins. 272 21

Four cAMP-independent receptor protein mutants (designated CRP* mutants) isolated previously are able to activate in vivo gene transcription in the absence of cAMP and their activity can be enhanced by cAMP or cGMP. One of the four mutant proteins, CRP*598 (Arg-142 to His, Ala-144 to Thr), has been characterized with regard to its conformational properties and ability to bind to and support abortive initiation from the lac promoter. In the absence of cGMP, CRP*598 shows a more open conformation than CRP, as indicated by its sensitivity to proteolytic attack and 5,5'-dithiobis(2-nitrobenzoic acid)-mediated subunit crosslinking. Binding of wild-type CRP to its site on the lac promoter and activation of abortive initiation by RNA polymerase on this promoter are effected by cAMP but not by cGMP. CRP*598 can activate lacP+-directed abortive initiation in the presence of cAMP and less efficiently in the presence of cGMP or in the absence of cyclic nucleotide. DNase I protection ("foot-printing") indicates that cAMP-CRP* binds to its site on the lac promoter whereas unliganded CRP* and cGMP-CRP* form a stable complex with the [32P]lacP+ fragment only in the presence of RNA polymerase, showing cooperative binding of two heterologous proteins. This cooperative binding provides strong evidence for a contact between CRP and RNA polymerase for activation of transcription. Although cGMP binds to CRP, it cannot replace cAMP in effecting the requisite conformational transition necessary for site-specific promoter binding. In contrast, the weakly active unliganded CRP*598 can be shifted to a functional state not only by cAMP but also by cGMP and RNA polymerase.
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PMID:Cooperative DNA binding of heterologous proteins: evidence for contact between the cyclic AMP receptor protein and RNA polymerase. 283 57

To determine whether chloroplast RNA polymerase will accurately terminate transcription in vitro, we have fused the spinach chloroplast rbcL promoter to the 3' end of the rbcL gene as well as to various factor independent transcription terminators from E. coli. Transcription of the rbcL minigene did not result in production of the expected 265 nucleotide RNA. However, the spinach chloroplast RNA polymerase did terminate transcription with varying efficiency at the thra, rrnB, rrnC and gene 32 terminators. The most efficient transcription termination was observed for the threonine attenuator. For each of the prokaryotic terminators, the chloroplast enzyme ceased transcription at essentially the same position as the E. coli RNA polymerase. These data indicate that the transcription termination process in chloroplasts has some features in common with the mechanism used in prokaryotes.
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PMID:Recognition of prokaryotic transcription terminators by spinach chloroplast RNA polymerase. 284 17

Subunit IIa of mammalian RNA polymerase II contains at its C terminus 52 tandem repeats of the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain is unmodified in RNA polymerase IIA, extensively phosphorylated in RNA polymerase IIO, and absent from RNA polymerase IIB. In an effort to define the role of the C-terminal domain, we have measured the transcriptional activity of purified RNA polymerases IIO, IIA, and IIB. The ability of each polymerase subspecies to transcribe the major late promoter of adenovirus-2 was examined in a polymerase-dependent transcription system reconstituted from partially purified transcription factors. RNA polymerases IIO, IIA, and IIB are all capable of initiating specific transcripts from this promoter. The transcriptional activity was determined as a function of the concentration of RNA polymerase II, template DNA, and each of the essential general transcription factors. The transcriptional activities of RNA polymerases IIA and IIB were comparable and consistently greater than that of RNA polymerase IIO when assayed under the conditions described here. The kinetics of transcript formation is similar except that RNA polymerase IIO has a more pronounced lag. These results show that the C-terminal domain of subunit IIa is not essential for the accurate initiation of transcripts from the major late promoter of adenovirus-2 and that the effect of the C-terminal domain is not likely mediated by the general transcription factors required for the expression of class II genes.
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PMID:The major late promoter of adenovirus-2 is accurately transcribed by RNA polymerases IIO, IIA, and IIB. 291 48

A protein kinase that phosphorylates Lys(Tyr-Ser-Pro-Thr-Ser-Pro-Ser)4, a synthetic peptide homologous to the evolutionarily-conserved, tandemly-repeated heptapeptide sequence at the C-terminus of the large subunit of eukaryotic RNA polymerase II, has been detected in HeLa cell extracts and chromatographic fractions therefrom. The enzyme, which phosphorylates serine principally, can be distinguished from previously described major protein kinases which phosphorylate the peptide poorly, if at all. It is inhibited by the nucleoside analog, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Results suggest that human placental RNA polymerase II is phosphorylated at the C-terminus of the large subunit by the partially-purified protein kinase and that the phosphorylation is also sensitive to the nucleoside analog.
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PMID:5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits a HeLa protein kinase that phosphorylates an RNA polymerase II-derived peptide. 293 May 26


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