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 protozoa Tetrahymena excretes a small peptide complex with an Mr of about 5,000. The peptide inhibits transcription by reducing the activity of the RNA polymerase. We have purified and partially characterized the peptide complex. It contains two peptide chains of apparent Mr 2,300 and 2,600, respectively. Magnesium ions in connection with the SH groups of cysteine play a role in holding together the two chains of the intact complex.
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PMID:Purification and partial characterization of a transcription-inhibitory peptide from Tetrahymena. 271 97

srnB is an F-plasmid encoded gene, otherwise silent, whose expression is induced by added rifampicin, leading to the release of cellular Mg2+ and degradation of stable RNA. In the absence of rifampicin, transcripts from the srnB gene were relatively short. S1 nuclease mapping revealed that the short mRNA species terminated within the leader, at the 3' end of a potential stem-and-loop structure. A deletion in the stem-loop resulted in constitutive synthesis of the mRNA that extended beyond the termination site into the structural gene. Even with the wild-type gene, transcription continued beyond the terminator sequence in the presence of added rifampicin. Most of the transcripts synthesized in the presence of rifampicin were long enough to encode the srnB protein. We hypothesize from these results that RNA polymerase associated with rifampicin can read through the terminator to induce srnB expression.
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PMID:Transcriptional regulation of F plasmid gene srnB: rifampicin-promoted in vitro readthrough of a terminator in the leader region. 274 21

We have used NMR to study the structure of the yeast tRNA(Phe) sequence which was synthesized by using T7 RNA polymerase. Many resonances in the imino 1H- spectrum of the transcript have been assigned, including those of several tertiary interactions. When the Mg2+ concentration is high, the transcript appears to fold normally, and the spectral features of the transcript resemble those of tRNA(Phe). The transcript has been shown to be aminoacylated with kinetics similar to the modified tRNA(Phe) [Sampson, J. R., & Uhlenbeck, O. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1033-1037], suggesting that the structure of the two molecules must be similar. In the absence of Mg2+ or at [tRNA]:[Mg2+] ratios less than 0.2, the transcript does not adopt the native structure, as shown by both chemical shifts and NOE patterns. In these low Mg2+ conditions, a second GU base pair is found, suggesting a structural rearrangement of the transcript. NMR data indicate that the structure of a mutant having G20 changed to U20 is nearly identical with that of the normal sequence, suggesting that the low aminoacylation activity of this variant is not due to a substantially different conformation.
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PMID:Structure of an unmodified tRNA molecule. 277 36

Three overlapping RNA fragments containing the pseudoknot, as found in the tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA, have been isolated and purified. Site-directed cleavage of TYMV RNA by RNase H, followed by ammonium sulphate precipitation and ion-exchange HPLC, yielded a pure preparation of a 3'-terminal, 112-nucleotide TYMV RNA fragment. Transcription of TYMV cDNA by T7 RNA polymerase, resulted in the isolation of an 88-nucleotide fragment. Finally, a 44-nucleotide fragment containing the TYMV RNA pseudoknot and strongly resembling the aminoacyl acceptor arm of the viral RNA was also synthesised using T7 RNA polymerase. The three fragments were isolated in milligram amounts and used for biochemical structure mapping, ultraviolet melting studies and NMR spectroscopy. Chemical modification with diethyl pyrocarbonate and sodium bisulphite and enzymatic digestion with RNase T1 confirmed the presence of the pseudoknot in the 44-nucleotide fragment. Also the analogue of the T-stem and T-loop of the tRNA-like structure of TYMV RNA was found. The results of modification at various temperatures in Mg2+-containing buffers were in general agreement with optical melting studies. Ultraviolet melting analysis of the longer fragments revealed their greater complexity and the results appear similar to those obtained for some tRNA species. To obtain direct biophysical evidence for base-pairing and stacking interactions in the pseudoknot, NMR studies were initiated. The first proton-NMR spectra ever obtained for plant viral RNA fragments are presented. NMR spectra were recorded at various buffer conditions and at various temperatures. The spectra for the 112-nucleotide and 88-nucleotide fragment are too complicated to be solved at present. In the case of the 44-nucleotide fragment, however, the imino proton resonances are well separated and this system turns out to be most promising for structural studies.
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PMID:Biochemical and biophysical analysis of pseudoknot-containing RNA fragments. Melting studies and NMR spectroscopy. 277 53

The fidelity of RNA replication by the poliovirus-RNA-dependent RNA polymerase was examined by copying homopolymeric RNA templates in vitro. The poliovirus RNA polymerase was extensively purified and used to copy poly(A), poly(C), or poly(I) templates with equimolar concentrations of noncomplementary and complementary ribonucleotides. The error frequency was expressed as the amount of a noncomplementary nucleotide incorporated divided by the total amount of complementary and noncomplementary nucleotide incorporated. The polymerase error frequencies were very high and ranged from 7 x 10(-4) to 5.4 x 10(-3), depending on the specific reaction conditions. There were no significant differences among the error frequencies obtained with different noncomplementary nucleotide substrates on a given template or between the values determined on two different templates for a specific noncomplementary substrate. The activity of the polymerase on poly(U) and poly(G) was too low to measure error frequencies on these templates. A fivefold increase in the error frequency was observed when the reaction conditions were changed from 3.0 mM Mg2+ (pH 7.0) to 7.0 mM Mg2+ (pH 8.0). This increase in the error frequency correlates with an eightfold increase in the elongation rate that was observed under the same conditions in a previous study.
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PMID:Direct measurement of the poliovirus RNA polymerase error frequency in vitro. 282 15

The pseudorabies virus immediate early (IE) protein, partially purified from infected HeLa cells, stimulated transcription initiation by RNA polymerase II and associated factors in HeLa nuclear extracts. This stimulation was maximal at low template concentrations, where the basal level of transcription was also low. In an analysis of the limitations on transcription under these conditions, it was found that transcription could be increased drastically not only by IE addition but also by (1) the addition of nonpromoter-containing DNA, which titrated nonspecific DNA-binding proteins in the crude nuclear extract, and (2) preincubation of the template with either the nuclear extract (in the absence of Mg2+) or with the TATA box-binding factor, TFIID. These results suggest that in the absence of IE, nonspecific DNA-binding proteins competed with TFIID for binding to the promoter, thus making TFIID: promoter interactions limiting for transcription. The stimulation of transcription effected by IE was essentially the same as that observed following preassociation of TFIID with the template or by titration of nonspecific DNA-binding proteins. Moreover, the presence of IE under the latter conditions did not stimulate transcription further. These observations strongly suggest that all of these manipulations affected the same limiting step and, thus, that IE accentuated the rate or extent of formation of a preinitiation complex involving the TATA factor, rather than subsequent initiation or elongation steps.
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PMID:The pseudorabies immediate early protein stimulates in vitro transcription by facilitating TFIID: promoter interactions. 283 79

RNA polymerase I preparations purified from a rat hepatoma contained DNA topoisomerase activity. The DNA topoisomerase associated with the polymerase had an Mr of 110,000, required Mg2+ but not ATP, and was recognized by anti-topoisomerase I antibodies. When added to RNA polymerase I preparations containing topoisomerase activity, anti-topoisomerase I antibodies were able to inhibit the DNA relaxing activity of the preparation as well as RNA synthesis in vitro. RNA polymerase II prepared by analogous procedures did not contain topoisomerase activity and was not recognized by the antibodies. The topoisomerase I: polymerase I complex was reversibly dissociated by column chromatography on Sephacryl S200 in the presence of 0.25 M (NH4)2SO4. Topoisomerase I was immunolocalized in the transcriptionally active ribosomal gene complex containing RNA polymerase I in situ. These data indicate that topoisomerase I and RNA polymerase I are tightly complexed both in vivo and in vitro, and suggest a role for DNA topoisomerase I in the transcription of ribosomal genes.
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PMID:Association of DNA topoisomerase I and RNA polymerase I: a possible role for topoisomerase I in ribosomal gene transcription. 285 18

DNA-dependent ATPase IV has been purified to near homogeneity from the Novikoff rat hepatoma. The enzyme is devoid of DNA polymerase, RNA polymerase, exonuclease, endonuclease, phosphomonoesterase, 3'- or 5'-phosphodiesterase, polynucleotide kinase, protein kinase, topoisomerase, helicase or DNA reannealing activities at a detection level of 10(-5) to 10(-7) relative to the ATPase activity. The enzyme is a monomer of Mr 110,000, has a sedimentation coefficient of 5.9 S, a Stokes radius of 40 A and a frictional coefficient of 1.32. In the presence of Mg2+ ion and a polynucleotide effector, ATPase IV hydrolyzes either ATP or dATP to the nucleoside diphosphate plus Pi. Other ribo- or deoxyribonucleoside triphosphates are not substrates. ATPase IV utilizes double-stranded DNA and single-stranded DNA as effector; however, it does not utilize poly(dT). The Km for dsDNA or ssDNA is 2.2 microM (nucleotide). A variety of ATP analogues were found to be competitive inhibitors of ATPase IV.
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PMID:Purification and enzymological characterization of DNA-dependent ATPase IV from the Novikoff hepatoma. 296 5

The kinetics of interaction of PPi and its diphosphonic analog, methylenediphosphonic acid (MDPA), with nucleoside triphosphates, DNA and Mg2+ binding sites of DNA-dependent RNA polymerase II from calf thymus was investigated. The values of apparent Km in the NTP polymerization reaction for ATP and CTP equal to 2.7 X 10(-4) and 1.8 X 10(-4) M, respectively, were determined. It was shown that MDPA and PPi competitively inhibited the RNA polymerase reaction with respect to nucleoside triphosphate. The inhibition constants (Ki) of ATP and CTP incorporation for MDPA were 2.2 X 10(-4) and 3.3 X 10(-4) M, respectively, while those of the nucleoside triphosphate incorporation for PPi were equal to 1.4 X 10(-4) and 2.0 X 10(-4) M, respectively. MDPA and PPi were incompetitive inhibitors of template (DNA) and Mn2+. A possible mechanism of inhibition of the RNA polymerase reaction by MDPA is proposed.
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PMID:[Kinetics of the interaction of methylene diphosphonic acid and inorganic pyrophosphate with DNA-dependent RNA-polymerase from calf thymus]. 298 49

Extracts of Xenopus laevis oocytes are able to assemble minichromosomes in vitro when they are supplemented with ATP and Mg2+. We have followed the time course of in vitro DNA supercoiling and transcription of polyoma virus closed circular DNA. The transcriptional activity increased with the assembly time of chromatin in the presence of both ATP and Mg2+, but only residual activity was detected in the absence of either of them. We also found that polyoma DNA as well as 5S RNA gene transcription were carried out mainly by an RNA polymerase III. On the other hand, polyoma RNA and 5S RNA synthesis were inhibited by novobiocin in a way that suggested the requirement of a DNA topoisomerase II or DNA gyrase activity for the initiation of transcription.
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PMID:In vitro transcription by Xenopus oocytes RNA polymerase III requires a DNA topoisomerase II activity. 300 12


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