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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)

An affinity-purified antibody raised against the fifth largest subunit of cauliflower (Brassica oleracea) RNA polymerase II was used to screen an expression library and isolate an Arabidopsis thaliana cDNA clone. This cDNA clone was used to isolate a soybean (Glycine max) cDNA clone, and both clones were sequenced. The open reading frames contain 176 amino acids and predict polypeptides of 19.5 and 19.6 kDa for Arabidopsis and soybean, respectively. The amino acid sequences of the Arabidopsis and soybean polypeptides are 91.5% identical. The fifth largest subunit in plant RNA polymerase II is present at unit stoichiometry in purified enzyme and does not dissociate from the holoenzyme during nondenaturing polyacrylamide gel electrophoresis. The gene encoding the 19.5-kDa subunit has been isolated and sequenced from Arabidopsis. The gene is single copy and contains five introns. The size of the mRNA encoding this RNA polymerase II subunit in Arabidopsis and soybean is approximately 1 kilobase. None of the published yeast or animal RNA polymerase subunit sequences show similarity to the fifth largest subunit in plants.
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PMID:Sequence of the fifth largest subunit of RNA polymerase II from plants. 142 63

RNA polymerase II is the core of the complex apparatus that is responsible for the regulated synthesis of mRNA. A comprehensive knowledge of RNA polymerase II is essential to our understanding of the molecular mechanisms through which a variety of transcription factors regulate eukaryotic gene expression. The recent cloning of genes for all ten subunits of yeast RNA polymerase II has revealed intriguing similarities and differences between the eukaryotic RNA polymerase and its simpler prokaryotic counterpart. Epitope tagging and other experiments made possible by the cloning of these genes have provided a clearer picture of RNA polymerase II subunit composition, stoichiometry and function, and set the stage for further investigating the dialogue between RNA polymerase II and transcription factors.
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PMID:RNA polymerase II: subunit structure and function. 170 May 3

RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides. The immunopurified enzyme catalyzed alpha-amanitin-sensitive RNA synthesis in vitro. The 10 polypeptides that immunoprecipitated were identical in size and number to those previously described for RNA polymerase II purified by conventional column chromatography. The relative stoichiometry of the subunits was deduced from knowledge of the sequence of the subunits and from the extent of labeling with [35S]methionine. Immunoprecipitation from 32P-labeled cell extracts revealed that three of the subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo. Phosphorylated and unphosphorylated forms of RPB1 could be distinguished; approximately half of the RNA polymerase II molecules contained a phosphorylated RPB1 subunit. These results more precisely define the subunit composition and phosphorylation of a eucaryotic RNA polymerase II enzyme.
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PMID:RNA polymerase II subunit composition, stoichiometry, and phosphorylation. 218 13

The Saccharomyces cerevisiae gene encoding the smallest RNA polymerase II subunit, RPB10, was isolated and sequenced. The gene for this subunit is present in single copy and maps to chromosome XV, where two other yeast RNA polymerase II subunits, RPB2 and RPB8, reside. The RPB10 sequence predicts a protein only 46 amino acids in length with a molecular mass of 5400 daltons. Sporulation and tetrad analysis of diploid cells containing one copy of the RPB10 gene and one copy of HIS3 in place of the RPB10 gene revealed that the RPB10 subunit is essential for viability.
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PMID:RNA polymerase II subunit RPB10 is essential for yeast cell viability. 850 44

RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range.
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PMID:RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth. 267 72

To improve our understanding of RNA polymerase II, the gene that encodes its third-largest subunit, RPB3, was isolated from a lambda gt11 DNA library by using antibody probes. The RPB3 DNA sequence predicts a 318-amino-acid protein whose sequence was confirmed, in part, by microsequence analysis of the gel-purified RNA polymerase II subunit. RPB3 was found to be an essential single-copy gene that is tightly linked to HIS6 on chromosome IX. An RPB3 temperature-sensitive mutant that arrested growth after three to four generations at the restrictive temperature was isolated. When the mutant was shifted to the restrictive temperature, RNA polymerase II could no longer assemble, previously assembled functional enzyme was depleted, and mRNA levels were consequently reduced. These results demonstrate that RPB3 is an essential component of the mRNA transcription apparatus. Finally, the RPB3 protein is similar in sequence and length to RPC5, a subunit common to RNA polymerases I and III, suggesting that these subunits may play similar roles in RNA polymerases I, II, and III.
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PMID:RNA polymerase II subunit RPB3 is an essential component of the mRNA transcription apparatus. 268 62

Genomic sequences for the large subunit of human RNA polymerase II corresponding to a part of the fifth exon were inserted into an expression vector at the carboxy-terminal end of the beta-galactosidase gene. The in-frame construct produced a 125-kilodalton fusion protein, containing approximately 10 kilodaltons of the large subunit of RNA polymerase II and 116 kilodaltons of beta-galactosidase. The purified bacterially produced fusion protein inhibited specific transcription from the adenovirus type 2 major late promoter, while beta-galactosidase had no effect. This effect of the fusion protein was during RNA elongation, not at the level of initiation, resembling the faithfully initiated but incomplete transcripts produced with purified factors in the absence of SII. Similarly, monoclonal antibody 2-7B, which reacts with the RNA polymerase II region represented in the fusion protein, inhibited specific transcription at the level of elongation in a whole-cell extract. Both monoclonal antibody 2-7B and the fusion protein, although unable to inhibit purified RNA polymerase II in a nonspecific transcription assay, selectively blocked the stimulation elicited by transcription elongation factor SII on the activity of the purified enzyme in vitro. This suggests that the fusion protein traps the SII in nonstimulatory interactions and that antibody 2-7B inhibits SII binding to RNA polymerase II. Thus, this suggests that an SII-binding contact required for specific RNA elongation resides within the fifth exon region of the largest RNA polymerase II subunit.
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PMID:Transcription elongation factor SII interacts with a domain of the large subunit of human RNA polymerase II. 314 7

Three subspecies of RNA polymerase II, designated IIO, IIA, and IIB, have been described in calf thymus and shown to differ in the apparent molecular weight of their largest subunits, designated IIo, IIa, and IIb, respectively. The objective of this study was to develop a procedure for the purification of RNA polymerase IIO. This form of the enzyme predominates in vivo and is responsible for the transcription of most cellular genes. RNA polymerase II is solubilized from isolated calf thymus nuclei in the presence of high concentrations of chelators, precipitated with polyethyleneimine, extracted with salt, and precipitated with (NH4)2SO4. The solubilized enzyme is resolved from factors that destabilize RNA polymerase IIO by chromatography on heparin-Sepharose CL-4B and DE52. RNA polymerase IIO is then partially resolved from RNA polymerases IIA and IIB by chromatography on DEAE-5PW and further purified by chromatography on Phenyl-Superose and Mono Q. RNA polymerase IIO was purified 1000-fold from the polyethyleneimine eluate resulting in about 130 micrograms of RNA polymerase IIO from 300 g of calf thymus. The specific activity of RNA polymerase IIO, in nonselective assays using calf thymus DNA as template, is 440 units/mg and not significantly different from that of RNA polymerases IIA and IIB. The similar transcriptional activities in nonselective assays suggest that the C-terminal domain of the largest RNA polymerase II subunit does not play a major role in the elongation phase of the reaction when deproteinized DNA serves as template. The small subunits of RNA polymerase IIO are indistinguishable from those of RNA polymerases IIA and IIB.
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PMID:Purification of RNA polymerase IIO from calf thymus. 319 3

Evidence is presented that isoproterenol treatment of rat C6 glioma cells, under conditions that increase glioma cell cAMP levels, causes the phosphorylative modification of several RNA polymerase II subunits. RNA polymerase II in control and isoproterenol-stimulated 32Pi-labeled confluent glioma cells was immunoprecipitated from ribonuclease-treated nuclear extracts with hen anti-calf RNA polymerase II antiserum conjugated to Sepharose. The immunoprecipitated RNA polymerase II was analyzed for 32P-labeled subunits by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Using this technique, we have shown that isoproterenol causes a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 214,000, 180,000, 140,000, 35,000, 28,000, and 16,500 daltons. Phosphate incorporation occurred exclusively on serine in all of the six subunits. About 0.5-2 mol of phosphate/mol of RNA polymerase II subunit were incorporated. Dibutyryl cAMP (10(-3)M) mimics the stimulatory action of isoproterenol and mediates increased phosphate incorporation into the six subunits. (RS)-propranolol (10(-4)M) prevents the isoproterenol-mediated phosphorylative changes. These data indicate that isoproterenol, via cAMP, mediates a transient structural modification of RNA polymerase II subunits in rat C6 glioma cells which may possibly lead to a modulation of RNA polymerase II function(s).
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PMID:Phosphorylation of rat C6 glioma cell DNA-dependent RNA polymerase II in vivo. Identification of phosphorylated subunits and modulation of phosphorylation by isoproterenol and N6,O2'-dibutyryl cyclic AMP. 609 70

Antibodies raised against the 180-kDa subunit of cauliflower RNA polymerase II bind selectively to the largest subunit of RNA polymerase II purified from a variety of plant species. The selective binding of this antibody to the largest RNA polymerase II subunit has allowed us to probe for the size of this subunit in crude cell extracts, in fractions containing partially purified RNA polymerase II, and in isolated nuclei. Fractions containing RNA polymerase II were subjected to electrophoresis in the presence of sodium dodecyl sulfate, blotted onto nitrocellulose, and blots were probed with antibody. Immunoglobulin complexes were revealed with 125I-Protein A. Published purification procedures result in rapid conversion of a 220-kDa subunit to a 180-kDa polypeptide, but purification at high pH (pH 9.0) retards this proteolysis. RNA polymerase II associated with isolated nuclei is largely protected from proteolytic degradation, and a 240-kDa polypeptide as well as a 220-kDa polypeptide can be detected. These results suggest that the 180-kDa subunit of RNA polymerase II arises artificially during cell lysis and enzyme purification, and that even the 220-kDa polypeptide may be a degradation product of a 240-kDa polypeptide in plants.
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PMID:Size heterogeneity of the largest subunit of nuclear RNA polymerase II. An immunological analysis. 636 44


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