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 gene rpo35, encoding a subunit of the vaccinia virus DNA-dependent RNA polymerase, was identified, and its RNA and protein products were characterized. An Mr 35,000 polypeptide, which bound antibody to the purified RNA polymerase, was synthesized in reticulocyte lysates programmed with viral mRNA that hybridized to a 2,300-base pair segment of the viral genome. Determination of the sequence of the DNA segment revealed four potential protein coding regions, none of which had evident similarity to any described RNA polymerase subunit of prokaryotes or eukaryotes. One open reading frame that could encode a 35,400-Da protein was identified as rpo35 on the basis of mRNA hybridization, cell-free translation, and immunoprecipitation. The identification was confirmed by sequencing tryptic peptides of the authentic Mr 35,000 RNA polymerase subunit. Antiserum to the purified recombinant protein, expressed in bacteria, reacted specifically with a Mr 35,000 polypeptide that was detected starting 2 h after virus infection and that co-sedimented with RNA polymerase purified from virions. RNA analyses indicated that the 5'-end of an early transcript started 25 nucleotides upstream of rpo35, which is consistent with the location of an early promoter consensus sequence.
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PMID:Identification, sequence, and expression of the gene encoding a Mr 35,000 subunit of the vaccinia virus DNA-dependent RNA polymerase. 185 5

RNA polymerases I, II, and III share three subunits that are immunologically and biochemically indistinguishable. The Saccharomyces cerevisiae genes that encode these subunits (RPB5, RPB6, and RPB8) were isolated and sequenced, and their transcriptional start sites were deduced. RPB5 encodes a 25-kD protein, RPB6, an 18-kD protein, and RPB8, a 16-kD protein. These genes are single copy, reside on different chromosomes, and are essential for viability. The fact that the genes are single copy, corroborates previous evidence suggesting that each of the common subunits is identical in RNA polymerases I, II, and III. Furthermore, immunoprecipitation of RPB6 coprecipitates proteins whose sizes are consistent with RNA polymerase I, II, and III subunits. Sequence similarity between the yeast RPB5 protein and a previously characterized human RNA polymerase subunit demonstrates that the common subunits of the nuclear RNA polymerases are well conserved among eukaryotes. The presence of these conserved and essential subunits in all three nuclear RNA polymerases and the absence of recognizable sequence motifs for DNA and nucleoside triphosphate-binding indicate that the common subunits do not have a catalytic role but are important for a function shared by the RNA polymerases such as transcriptional efficiency, nuclear localization, enzyme stability, or coordinate regulation of rRNA, mRNA, and tRNA synthesis.
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PMID:Subunits shared by eukaryotic nuclear RNA polymerases. 218 66

Antibody was raised against purified vaccinia virus RNA polymerase and used to screen a recombinant vaccinia virus-lambda gt11 library. The DNA from several immunopositive clones was shown by Southern hybridization to originate from the vaccinia virus HindIII E fragment. The nucleotide sequence of the RNA polymerase subunit gene predicts a polypeptide 287 amino acids in length and 30,000 daltons in mass. An early transcript with a 5' terminus just upstream of the putative initiation codon was identified by S1 nuclease protection and primer extension analyses, demonstrating that this RNA polymerase subunit is expressed as an early viral gene product. The RNA polymerase subunit was synthesized by a bacterial expression vector to demonstrate that it corresponds to the previously described 37,000-dalton RNA polymerase subunit.
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PMID:Vaccinia virus gene encoding a 30-kilodalton subunit of the viral DNA-dependent RNA polymerase. 221 20

The DNA-dependent RNA polymerase of vaccinia virus contains 8 to 10 virus-encoded polypeptides. We have mapped the gene encoding an 18-kilodalton RNA polymerase subunit to D7R, the seventh open reading frame of the HindIII D genomic subfragment. Localization of this gene was achieved by using antibody to the purified RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments. The identification was confirmed by translation of D7R transcripts made in vitro with bacteriophage T7 RNA polymerase. The phenotypes of two previously isolated conditionally lethal temperature-sensitive mutants that map to D7R (J. Seto, L. M. Celenza, R. C. Condit, and E. G. Niles, Virology 160:110-119, 1987) are consistent with an essential role of this subunit in late transcription. This polymerase gene, designated rpo18, predicts a polypeptide of 161 amino acids with a molecular mass of 17,892. The rpo18 gene is transcribed early in infection, even though the 5'-TAAATG-3' motif, which is conserved among many genes of the late class, is present near the RNA start site. Characterization of the 5' end of the early transcript by several different methods, including cDNA cloning, revealed a poly(A) leader with up to 14 adenylate residues, whereas only 3 are present in the corresponding location of the DNA template. Similar but somewhat longer poly(A) leaders have previously been observed in mRNAs of late genes. We noted a TAAATG motif near the initiation site of several other early genes, including the viral DNA polymerase, and carried out additional experiments to demonstrate that their early transcripts also have 5' poly(A) leaders. Thus, formation of the poly(A) leader is not exclusively a late function but apparently depends on sequences around the transcription initiation site.
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PMID:Identification of the vaccinia virus gene encoding an 18-kilodalton subunit of RNA polymerase and demonstration of a 5' poly(A) leader on its early transcript. 233 25

The largest subunit of mammalian RNA polymerase II contains at its C terminus an unusual domain consisting of multiple tandem repeats of the seven-amino acid consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. This domain is unphosphorylated in RNA polymerase IIA and extensively phosphorylated in RNA polymerase IIO. To investigate the role of the C-terminal domain and the functional significance of its phosphorylation, changes in the level of phosphorylation were followed as a function of the position of RNA polymerase II in the transcription cycle. Complexes were formed with 32P-labeled RNA polymerase IIA and separated from the free polymerase by gel filtration. The phosphorylation state of the RNA polymerase II largest subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results indicate that RNA polymerase IIA interacts with the template-committed complex to form a stable preinitiation complex. RNA polymerase IIA associated with such complexes is converted to RNA polymerase IIO in the presence of ATP prior to the formation of the first phosphodiester bond. Furthermore, the observation that purified preinitiation complexes can catalyze the conversion of RNA polymerase IIA to IIO indicates that the protein kinase(s) responsible for phosphorylation of the C-terminal domain is a component of such complexes. The concentration of ATP required for the phosphorylation of RNA polymerase II associated with the preinitiation complex is two to three orders of magnitude lower than that required for the conversion of RNA polymerase IIA to IIO free in solution. These results support the idea that phosphorylation of the C-terminal domain of RNA polymerase subunit IIa occurs subsequent to the association of enzyme with the promoter and prior to the initiation of transcription.
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PMID:Phosphorylation of RNA polymerase IIA occurs subsequent to interaction with the promoter and before the initiation of transcription. 237 91

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.
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PMID:Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor. 239 97

The genes for the three large subunits A, B and C, of the DNA-dependent RNA polymerase of the archaebacterium Sulfolobus acidocaldarius DSM 639, were identified and characterized. The three genes follow each other immediately in the order B-A-C, which corresponds to that found in the rpoBC operon of the Escherichia coli genome. The transcription products formed in vivo were studied by Northern analysis and the start-points were determined by S1-nuclease mapping and primer directed extension analysis. The three RNA polymerase subunit genes were co-transcribed together with an open reading frame (ORF) of 88 amino acid residues length situated immediately upstream of the B gene and two ORFs of 104 and 130 amino acid residues following the C gene (together 8500 nucleotides). The following ORF, encoding a protein of 118 amino acids homologous to the ribosomal protein S12 of E. coli, was weakly transcribed with the large co-transcript and strongly from an own promoter. The derived amino acid sequence of the B-subunit was found to be homologous to the B- (second largest) subunits of the eukaryotic nuclear polymerases I, II and III and to the eubacterial beta-subunit. The combined A + C-subunits correspond to the A- (largest) subunits of the eukaryotic RNA polymerases I, II and III and to the eubacterial beta'-subunit. The amino acid sequence similarity of the Sulfolobus subunits to the eukaryotic components is clearly higher than to the E. coli subunit.
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PMID:Organization and nucleotide sequence of the genes encoding the large subunits A, B and C of the DNA-dependent RNA polymerase of the archaebacterium Sulfolobus acidocaldarius. 250 56

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

We have studied the factors that affect transcription termination in vitro at the tR2 terminator of bacteriophage lambda and at the T1 terminator of the Escherichia coli rrnB operon. Termination efficiency at both of these sites is enhanced by the E. coli nusA protein, giving final efficiencies of termination in vitro comparable to those estimated in vivo. Transcripts terminated in the presence of nusA protein are all released from the RNA polymerase complex, indicating that a complete termination reaction is involved, rather than simply induction of a long pause at the terminator. The termination factor activity of the nusA protein does not depend on the presence of rho protein and is not detectably enhanced by that factor. Thus, the nusA protein appears to play a pleiotropic role in E. coli transcription, serving as an antitermination factor, RNA polymerase subunit and true termination factor for some terminator sites.
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PMID:nusA protein of Escherichia coli is an efficient transcription termination factor for certain terminator sites. 282 Dec 82

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


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