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)

Two DNA-dependent RNA polymerases have been purified from homogenates of maize leaves by a relatively rapid procedure involving Sepharose 4B and DEAE-cellulose chromatography followed by resolution of two RNA polymerasies on phosphocellulose. The RNA polymerase eluting from phosphocellulose at 0.11 M (NH4)2SO4 is inhibited strongly by low levels of alpha-amanitin and possesses catalytic properties and polypeptide subunits like those of maize nuclear RNA polymerase II. The RNA polymerase eluting from phosphocellulose at 0.15 M (NH4)2SO4 resembles the RNA polymerase solubilized from isolated maize chloroplasts in many ways. This enzyme and that isolated from chloroplasts are resistant to alpha-amanitin and rifamycin-SV at high concentrations. Both RNA polymerases have virtually the same Mn2+ and Mg2+ optima, Mg2+/Mn2+ activity ratios, (NH4)2SO4 sensitivity, kinetics of UMP incorporation, and temperature optima. Electrophoresis of this phosphocellulose-purified RNA polymerase on denaturing polyacrylamide slab gels reveals 14 heavily stainable polypeptides that are identical in number and molecular mass to those from chloroplast RNA polymerase. Moreover, two-dimensional tryptic maps of the 14 polypeptides from the phosphocellulose-purified RNA polymerase are very similar to the maps of corresponding polypeptides from chloroplast RNA polymerase. Using this method, relatively large quantities (0.5 mg/kg leaves) of a form of chloroplast RNA polymerase can be prepared in a few days.
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PMID:A facile procedure for purifying maize chloroplast RNA polymerase from whole cell homogenates. 740 81

Treatment of Pseudomonas phaseolicola double-stranded RNA bacteriophage phi 6 with sodium deoxycholate converted the virions to nucleocapsids, which had in vitro RNA polymerase activity. The incorporation of [3H]UMP continued for at least 7 h. The initial incorporation was detected as intermediate RNA. Radioactivity was chased first into three segments of double-stranded RNA, and then into small, medium, and large species of single-stranded RNA successively via the intermediate RNA. Several copies of single-stranded RNA at least were synthesized from a template. The RNA synthesis clearly took place by a semi-conservative mechanism with respect to templates. That is, 5-bromo UTP was incorporated into one strand of double-stranded RNA to make a hybrid RNA of brominated and unbrominated strands. Furthermore, one strand of the 3H-labeled parental double-stranded RNA was shown to be released as single-stranded RNA.
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PMID:Semi-conservative transcription of double-stranded RNA catalyzed by bacteriophage phi 6 RNA polymerase. 746 97

We have examined the interaction between NusA and the nascent RNA in Escherichia coli transcription complexes on four different templates. Photocrosslinking CTP and UTP analogs were incorporated internally and at the 3' end of the RNA. Identical templates with and without boxA sequences were compared. We found that NusA did not contact the ten nucleotides nearest to the 3' end of the RNA in complexes containing RNA up to 20 nucleotides long. Longer RNA did crosslink to NusA with all four templates examined, however. We reported that RNA 80 nucleotides long from the bacteriophage T7 A1 promoter substituted in two RNA stem-loops with photocrosslinking UMP analogs did not crosslink to NusA, even though interaction between NusA and the transcription complex were demonstrated. Here, we report that when this same RNA is substituted at CMP residues, it does crosslink to NusA. Templates containing the E. coli ribosomal RNA promoter rrnG P2, with and without a boxA sequence downstream, were compared. Long RNAs from both crosslinked to NusA, and thus boxA RNA sequences are not required for interaction with NusA. NusA did not interact with the free RNA containing boxA once released from the transcription complex, nor did it interact with RNA in a binary complex containing only RNA polymerase and RNA, without the DNA template.
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PMID:NusA contacts nascent RNA in Escherichia coli transcription complexes. 753 48

The effects of NusA on the RNA polymerase contacts made by nucleotides at internal positions in the nascent RNA in Escherichia coli transcription complexes were analyzed by using the photocrosslinking nucleotide analog 5-[(4-azidophenacyl) thio]-UMP. It was placed at nucleotides between +6 and +15 in RNA transcribed from the phage lambda PR' promoter. Crosslinks of analog in these positions in RNAs which contained either 15, 28, 29, or 49 nt were examined. Contacts between the nascent RNA and proteins in the transcription complex were analyzed as the RNA was elongated, by placing the crosslinker nearest the 5' end of the RNA 10, 23, 24, or 44 nt away from the 3' end. The beta or beta' subunit of polymerase, and NusA when added, were contacted by RNA from 15 to 49 nt long. When the upstream crosslinker was 24 nt from the 3" end of the RNA (29-nt RNA), alpha was also contacted in the absence of NusA. The addition of NusA prevented RNA crosslinking to alpha. When the crosslinker was 44 nt from the 3' end (49-nt RNA), alpha crosslinks were still observed, but crosslinks to beta or beta' and NusA were greatly diminished. RNA crosslinking to alpha, and loss of this crosslink when NusA was added, was observed in the presence of NusB, NusE, and NusG and when transcription was carried out in the presence of an E. coli S100 cell extract. Peptide mapping localized the RNA interactions to the C-terminal domain of alpha.
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PMID:NusA interferes with interactions between the nascent RNA and the C-terminal domain of the alpha subunit of RNA polymerase in Escherichia coli transcription complexes. 753 40

A new photocrosslinking CTP analog that functioned as a substrate during transcription was synthesized and used to photoaffinity label E. coli and bacteriophage T7 RNA polymerases. This analog, 5-((4-azidophenacyl)thio) cytidine-5'-triphosphate (5-APAS-CTP) contains an aryl azide group approximately 10 A from the nucleotide base and specifically replaced CTP during synthesis of RNA by both polymerases. Analog was placed at the 3' end or internally within RNA. Both polymerases inefficiently incorporated two 5-APAS-CMP molecules sequentially, as was found for the related 5-APAS-UMP. Analog was placed at the 3' end of RNA in transcription complexes paused at the site of Q-modification of E. coli RNA polymerase, downstream of the lambda PR' promoter (+16), a pause that requires specific DNA sequences but no apparent RNA hairpin. Crosslinking was examined in the presence and absence of the NusA protein, which enhances the transcriptional pause at this site and is required for Q modification of the polymerase. Crosslinking of the 3' end of the RNA to NusA was not observed, consistent with our earlier results involving a NusA-enhanced pause site downstream from an RNA hairpin.
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PMID:Synthesis and characterization of a new photocrosslinking CTP analog and its use in photoaffinity labeling E. coli and T7 RNA polymerases. 768 33

A stable ternary transcription complex was formed with either wheat germ or yeast RNA polymerase II using a ribotrinucleotide primer (GpCpG) to initiate transcription on a short synthetic single-strand DNA template. The template was designed to limit the incorporation of a photoprobe S4-UMP (4-thio-UMP) to a unique position at the 3' terminus of the transcript. The resulting stable ternary transcription complex was photolyzed to cross-link the bound transcript ([32P]-labeled by the incorporation of [alpha-32P]CMP) with the protein domain at or near the active site. Separation of the protein components by electrophoresis in polyacrylamide gel containing SDS and analysis by autoradiography and silver staining revealed that for either enzyme only the largest subunit was [32P] labeled.
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PMID:Wheat germ and yeast RNA polymerase II: photoaffinity labeling by 4-thiouracil 5'-monophosphate positioned uniquely at the 3' end of an enzyme-bound [32P]-containing transcript. 844 67

Elongation factor SII (also known as TFIIS) is an RNA polymerase II binding protein that allows bypass of template arrest sites by activating a nascent RNA cleavage reaction. Here we show that SII contacts the 3'-end of nascent RNA within an RNA polymerase II elongation complex as detected by photoaffinity labeling. Photocross-linking was dependent upon the presence of SII, incorporation of 4-thio-UMP into RNA, and irradiation and was sensitive to treatment by RNase and proteinase. A transcriptionally active mutant of SII lacking the first 130 amino acids was also cross-linked to the nascent RNA, but SII from Saccharomyces cerevisiae, which is inactive in concert with mammalian RNA polymerase II, failed to become photoaffinity labeled. SII-RNA contact was not detected after a labeled oligoribonucleotide was released from the complex by nascent RNA cleavage, demonstrating that this interaction takes place between elongation complex-associated but not free RNA. This shows that the 3'-end of RNA is near the SII binding site on RNA polymerase II and suggests that SII may activate the intrinsic RNA hydrolysis activity by positioning the transcript in the enzyme's active site.
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PMID:Elongation factor SII contacts the 3'-end of RNA in the RNA polymerase II elongation complex. 879 87

Transcriptional attenuation of the pyrimidine biosynthetic (pyr) operon from Bacillus subtilis was reconstituted with an in vitro system that consisted of pyr DNA templates, B. subtilis RNA polymerase, four ribonucleoside triphosphates, and the purified B. subtilis PyrR regulatory protein. The templates used each specified one of the three known attenuation regions of the pyr operon. Runoff (read-though) and terminated transcripts of the predicted lengths were the only major products synthesized. Transcription of the template that specifies the 5' leader attenuation region of the operon was examined in detail. Termination of transcription at the attenuator was strongly promoted by the combination of PyrR plus UMP. The concentration of UMP required for half-maximal effect was 2.5 microM. UTP also promoted termination in the presence of PyrR, but concentrations 10-fold higher than UMP were required; UDP was only effective at 100 times the concentration of UMP. Other pyrimidine and purine metabolites tested did not affect termination. PRPP, which like UMP is a substrate for the uracil phosphoribosyltransferase activity of PyrR, antagonized UMP-dependent transcriptional termination, but uracil did not. Transcriptional attenuation by PyrR plus UMP was also demonstrated in vitro with templates from the other two pyr attenuation regions. The results strongly support the model for transcriptional regulation of the B. subtilis pyr operon previously proposed by R. J. Turner, Y. Lu, and R. L. Switzer (J. Bacteriol. 176:3708-3722, 1994).
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PMID:Transcriptional attenuation of the Bacillus subtilis pyr operon by the PyrR regulatory protein and uridine nucleotides in vitro. 895 3

Vaccinia virus RNA polymerase terminates transcription downstream of a UUUUUNU signal in the nascent RNA. Transduction of the RNA signal to the elongating polymerase requires a termination factor (vaccinia termination factor/capping enzyme) and is coupled to the hydrolysis of ATP. It was shown previously that incorporation of 5-bromouracil or 5-iodouracil within the UUUUUNU element abolishes termination by preventing factor-dependent release of the nascent chain from the polymerase elongation complex. Here, we report that termination is prevented by phosphorothioate substitution at UMP residues in the nascent RNA. In contrast, phosphorothioate substitution at AMP, CMP, and GMP nucleotides does not inhibit termination. Thus, the action of a eukaryotic termination factor entails recognition of the nucleotide bases and the phosphate groups of the target sequence in nascent RNA.
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PMID:Transcription termination by vaccinia RNA polymerase entails recognition of specific phosphates in the nascent RNA. 899 14

Little information is available on the effects of UVA (320-400 nm radiation) on transcription. We examined the effect of UVA on RNA synthesis in isolated chicken liver nuclei. Nuclei in air or nitrogen were irradiated with UVA, and the RNA synthesis induced by endogenous RNA polymerase was estimated under conditions in which little or no initiation occurs. Incorporation of [3H]UMP into the acid-insoluble fraction was used as the measure of RNA synthesis in the nuclei. In air the amount of synthesized RNA decreased with increasing UVA fluence. In contrast, in nitrogen UVA had little effect on RNA synthesis. Sodium azide and histidine, which effectively scavenge singlet oxygen (1O2) as well as hydroxyl radicals (.OH), protected the nuclei from inhibition of RNA synthesis; whereas, sodium formate and dimethyl sulfoxide, both of which much more effectively scavenge .OH than 1O2, had no protective effect. These findings provide a strong indication that 1O2 is involved in the inhibition of RNA synthesis. In addition, RNA polymerase II-dependent synthesis (in the nucleoplasm) was much more sensitive to UVA than RNA polymerase I-dependent synthesis (in the nucleolus).
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PMID:Effect of UVA on RNA synthesis in isolated chicken liver nuclei. 916 76


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