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 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 murine adipocyte lipid binding protein (ALBP) has been cloned into Escherichia coli, purified from expressing cultures, and its ligand binding and phosphorylation properties studied. In the cloning strategy, the recombinant, pT7-5 rALBP, was transformed into E. coli strain K38 harboring plasmid pGP1-2 which directs the synthesis of T7 RNA polymerase. Upon shifting the temperature from 30 to 42 degrees C to induce T7 RNA polymerase expression, the 14.6-kDa recombinant ALBP (rALBP) was expressed for approximately 2 h and accumulated to about 1% of total E. coli protein. The recombinant ALBP was soluble in E. coli extracts and resistant to bacterial proteolysis. A procedure for purifying rALBP was developed utilizing immuno-chemical detection based upon reactivity with anti-murine ALBP antiserum. A combination of acidic ammonium sulfate fractionation, gel permeation chromatography, and carboxymethyl ion-exchange high performance liquid chromatography separation was used to prepare homogeneous rALBP. Sequence analysis of rALBP indicated that the initiating methionine residue had been removed and the amino-terminal cysteine residue was not blocked. Purified rALBP exhibited stoichiometric, saturable binding of oleic acid (n = 1.0, K0.5 approximately 100 microM) and retinoic acid (n = 1.0, K0.5 approximately 170 microM). Incubation of rALBP with wheat germ agglutinin-purified insulin receptor, ATP, and 100 nM insulin resulted in a 5-fold stimulation of rALBP phosphorylation above the basal state. Kinetic analysis of rALBP phosphorylation by the 3T3-L1 insulin receptor kinase yielded a Michaelis constant (Km) of 50 microM and a maximal velocity of 1 mol of rALBP phosphorylated/min/mol insulin binding sites. Phosphoamino acid analysis indicated that phosphorylation occurred upon tyrosine. These results indicate that murine ALBP has been cloned and expressed in E. coli, purified to homogeneity, and is a substrate for the insulin receptor tyrosyl kinase in vitro.
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PMID:Cloning of murine adipocyte lipid binding protein in Escherichia coli. Its purification, ligand binding properties, and phosphorylation by the adipocyte insulin receptor. 268 57

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

The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.
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PMID:Sequence of the Bacillus subtilis glutamine synthetase gene region. 290 11

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

Purified eukaryotic nuclear RNA polymerase II consists of three subspecies that differ in the apparent molecular masses of their largest subunit, designated IIo, IIa, and IIb for polymerase species IIO, IIA, and IIB, respectively. Subunits IIo, IIa, and IIb are the products of a single gene. We present here the amino acid composition of calf thymus subunits IIa and IIb and the C-terminal amino acid sequence of subunit IIa (IIo) inferred from the nucleotide sequence of part of the mouse gene encoding this RNA polymerase subunit. The calculated amino acid composition of the peptide unique to subunit IIa indicates that subunit IIa contains a domain rich in serine, proline, threonine, and tyrosine. The sequence at the 3' end of the mouse RNA polymerase II largest subunit gene reveals that the C-terminal domain consists of 52 repeats of a seven amino acid block with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This sequence is also unusual in that it contains a high percentage of potential phosphorylation sites.
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PMID:A unique structure at the carboxyl terminus of the largest subunit of eukaryotic RNA polymerase II. 299 85

The genomic locus (RPII215) encoding the largest subunit of mouse RNA polymerase II has been cloned by low stringency hybridization to a Drosophila RPII215 probe. The mouse gene consists of 28 exons which span 30 kilobases. Analysis of the nucleotide and predicted protein sequences indicates that the protein is comprised of two domains. There is a 1500 residue amino-terminal domain which contains seven regions strikingly similar to those in the beta' subunit of Escherichia coli RNA polymerase, and a carboxyl-terminal domain comprised of 52 repeats of a 7-amino-acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Among the seven highly conserved regions are a strongly basic domain consistent with a DNA-binding site and a consensus sequence characteristic of a potential zinc-binding domain. The 5' upstream region contains three tandem sequences similar to binding sites for the transcription factor SP1. Two of the introns in this gene splice at donor GC dinucleotides as opposed to previously described invariant GT sites. The identification of regions which are highly conserved as compared with bacterial and yeast RNA polymerase and other regions which are unique to the mouse protein suggests which domains of RNA polymerase large subunits are involved in aspects of transcription common to both procaryotes and eucaryotes and which are characteristic of transcription in higher organisms.
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PMID:Cloning and sequence analysis of the mouse genomic locus encoding the largest subunit of RNA polymerase II. 303 94

Ts-phenotype of the E. coli rho-factor mutant rho 15 is suppressed by two rifampicin-resistance mutations, rhoB1019 resulting in a single amino acid substitution Val146----Phe and rhoB268 resulting in a single substitution Gln513----Leu in beta-subunit of the E. coli RNA polymerase. Rifampicin-resistance mutations rhoB255 (Asp516----Val), rhoB1016 (Asp516----Asn), rhoB1001 (His526----Tyr), rhoB1004 (Ser531----Phe), rhoB1005 (Pro564----Leu), and streptolydigin-resistance' mutation rhoB1018 (double substitution Gly544----Asp and Phe545----Ser) do not suppress the rho15 mutation.
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PMID:[Amino acid substitutions in the beta-subunit of RNA-polymerase from E. coli compensating for mutation-induced damage of the rho termination factors]. 305 19


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