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)

Ten min after infection of Xanthomonas campestris pv. oryzae by phage Xp10, a sharp decrease in the activity of the host RNA polymerase was observed. Host RNA polymerase from phage-infected and uninfected cells was purified, and their properties were compared. The enzyme from uninfected cells contained four polypeptides with Mr = 155,000, 155,000, 93,000, and 37,000, respectively, and assembled with a stoichiometry of alpha 2 beta beta' sigma. The enzyme from infected cells lacked the sigma-subunit. The enzyme from uninfected cells utilized Xp10 DNA and poly[d(A-T)] as templates, the enzyme from phage-infected cells failed to transcribe Xp10 DNA, but retained the ability to transcribe poly(A-T). The regions of the Xp10 genome transcribed by the two enzymes were also investigated. The enzyme from uninfected cells transcribed the leftmost 25-30% of the Xp10 genome. The enzyme from phage-infected cells also transcribed the same region, but the enzyme activity was very low. Other properties such as (a) the response to RNA polymerase inhibitors, (b) the effect of N-ethylmaleimide, (c) the requirement of Mg2+ and Mn2+, and (d) the optimum temperature and pH of the two enzymes were very similar.
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PMID:Loss of sigma-factor of RNA polymerase of Xanthomonas campestris pv. oryzae during phage Xp10 infection. 302 43

The splicing of a procaryotic precursor RNA transcribed from the T4 phage thymidylate synthase (td) gene with SP6 RNA polymerase was investigated in vitro. The intron excision-cyclization reaction increased progressively to 60 degrees C. Exon ligation, though barely detectable at the lower temperatures, was greatly enhanced at 60 degrees C. Both reactions required Mg2+. The addition of guanosine to the 5' end of an intron-exon II intermediate via a 3',5'-phosphodiester bond was essential for the ligation of exon I to exon II. The added guanosine and the first intron-encoded uridine are subsequently lost as a dinucleotide from the 5' end during cyclization of the linear form of the excised intron RNA. Exon ligation is intramolecular and occurs more readily in the nascent RNA molecule (cotranscriptionally) than in the finished transcript (posttranscriptionally). These data and the identification of various structural elements (P, Q, R, S, E, E') in the td intron that are found typically in eucaryotic class I introns firmly establish the td intron as the first example of class I intron of procaryotic origin.
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PMID:Mechanism and requirements of in vitro RNA splicing of the primary transcript from the T4 bacteriophage thymidylate synthase gene. 303 75

The interaction of Escherichia coli RNA polymerase with poly[d(A-T)] and poly[d-(I-C)] was studied by difference absorption spectroscopy at temperatures, from 5 to 45 degrees C in the absence and presence of Mg2+. The effect of KCl concentration, at a fixed temperature, was studied from 12.5 to 400 mM. Difference absorption experiments permitted calculation of the extent of DNA opening induced by RNA polymerase and estimation of the equilibrium constant associated with the isomerization from a closed to an open RNA polymerase-DNA complex. delta H0 and delta S0 for the closed-to-open transition with poly[d(A-T)] or poly[d(I-C)] complexed with RNA polymerase are significantly lower than the values associated with the helix-to-coil transition for the free polynucleotides. For the RNA polymerase complexes with poly[d(A-T)] and poly[d(I-C)] in 50 mM KCl, delta H0 approximately 15-16 kcal/mol (63-67 kJ/mol) and delta S0 approximately 50-57 cal/K per mol (209-239 J/K per mol). The presence of Mg2+ does not change these parameters appreciably for the RNA polymerase-poly[d(A-T)] complex, but for the RNA polymerase-poly[d(I-C)] complex in the presence of Mg2+, the delta H0 and delta S0 values are larger and temperature-dependent, with delta H0 approximately 22 kcal/mol (92 kJ/mol) and delta S0 approximately 72 cal/K per mol (approx. 300 J/K per mol) at 25 degrees C, and delta Cp0 approximately 2 kcal/K per mol (approx. 8.3 kJ/K per mol). The circular dichroism (CD) changes observed for helix opening induced by RNA polymerase are qualitatively consistent with the thermally induced changes observed for the free polynucleotides, supporting the difference absorption method. The salt-dependent studies indicate that two monovalent cations are released upon helix opening. For poly[d(A-T)], the temperature-dependence of enzyme activity correlates well with the helix opening, implying this step to be the rate-determining step. In the case of poly[d(I-C)], the same is not true, and so the rate-determining step must be a process subsequent to helix opening.
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PMID:Spectroscopic analysis of DNA base-pair opening by Escherichia coli RNA polymerase. Temperature and ionic strength effects. 304 7

8-oxy-GTP was obtained via reaction of GTP with ascorbic acid and addition of hydrogen peroxide. 8-oxy-GTP is recognized and displays substrate properties of UTP on substitution of 8-oxy-GTP for UTP in polynucleotide synthesis catalyzed by E. coli RNA polymerase on a poly[d(A-T)].poly[d(A-T)] template. Such incorporation does not take place at equimolar quantities of GTP and 8-Br-GTP. The incorporation of 8-oxy-GTP instead of UTP, is 2.5-3 times higher upon replacement of Mg2+ by Mn2+ ions. The dinucleotide ApU serving as an initiator rises the incorporation level of 8-oxy-GTP both for Mg2+ and Mn2+ ions. 8-oxy-GTP slightly inhibits poly[r(A-U)] synthesis, but UTP strongly inhibits the incorporation of 8-oxy-GTP. [alpha-32P] 8-oxy-GTP is incorporated mainly instead of UTP, but it can be incorporated also during the substitution of 8-oxy-GTP for ATP.
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PMID:[Display of 8-hydroxy-GTP substrate properties of UTP in the reaction of polynucleotide synthesis catalyzed by RNA-polymerase from Escherichia coli in the presence of poly[d(AT).d(AT)] template]. 305 96

DNA-dependent RNA polymerase was purified from Pseudomonas aeruginosa. The subunit structure was typical of other eubacterial RNA polymerases in having beta' (157,000), beta (148,000), sigma (87,000), and alpha 2 (45,000) subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was dependent on Mg2+, displaying optimal activity at 10 mM MgCl2. Ca2+ and Zn2+ could not replace MgCl2 in the assay system, while Mn2+, produced partial activity. KCl at concentrations greater than 10 mM inhibited enzyme activity. Optimal enzyme activity was observed at pH 8.5-9.0. The RNA polymerase was stable in 50% (w/v) glycerol at 4 degrees C for more than 3 months. Enzyme activity was inhibited in vitro by heparin, streptolydigin, streptovaracin, actinomycin D, and rifampicin.
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PMID:DNA-dependent RNA polymerase from Pseudomonas aeruginosa. 312 44

The effect of aflatoxin B1 on the DNA template and DNA-dependent RNA polymerases in buffalo liver was studied. Aflatoxin B1 inhibited both Mg2+- and Mn2+-activated RNA polymerases in a dose-dependent manner. At 10 micrograms the inhibition of both enzymes was almost complete. The inhibitory effect on the solubilized enzymes was higher than the chromatin-bound, suggesting a direct effect at the enzyme level. On the other hand, incubating DNA or deoxyribonucleoprotein (DNP) with 2 micrograms aflatoxin reduces its transcriptional capacity with a greater effect on the Mg2+-activated RNA polymerase than the Mn2+-activated enzyme. These results suggest that aflatoxin B1 inhibits in vitro transcription in buffalo liver at both enzyme and template levels.
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PMID:In vitro effect of aflatoxin B1 on the transcriptional activity of DNA template, chromatin and soluble DNA-dependent RNA polymerases in buffalo liver. 312 97

DNA-dependent RNA polymerase has been purified from gram-positive Lactobacillus acidophilus and found to be composed of 4 protein subunits, alpha, beta, beta', and sigma, with molecular weights of 40,000, 150,000, 135,000, and 45,000 kD, respectively, estimated on the basis of SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibits optimal activity in the presence of Mn2+, while Mg2+ shows only a slight effect. The L. acidophilus enzyme transcribes several Escherichia coli promoters examined so far, such as promoters of trp operon, lacUV5, and bla P3 from pBR322, whereas it lacks the ability to recognize bla P1 and tet P2 promoters from pBR322. Thus, the specificity of L. acidophilus RNA polymerase in recognizing the promoters is somehow different from that of the E. coli enzyme. By means of an in vitro transcription assay system for L. acidophilus RNA polymerase, 2 promoters have been identified in the DNA of an L. acidophilus cryptic plasmid (pRNL5). These promoters possess nucleotide sequences in the -10 region similar to the consensus sequence for the E. coli promoters.
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PMID:Characterization and promoter selectivity of Lactobacillus acidophilus RNA polymerase. 315 Jun 81

We report on the properties of a partially purified tRNA intron endonuclease from the archaebacterium Halobacterium volcanii. This enzyme is capable of precise excision of the 104-nucleotide intron from halo-bacterial pre-tRNA(Trp) substrates generated in vitro by T7 RNA polymerase transcription. The reaction requires divalent cations (Mg2+ or Ca2+) or spermidine, is inhibited by monovalent cations, and produces 5'-hydroxyl and 2',3'-cyclic phosphate termini. Unlike the universal substrate recognition properties characteristic of the eukaryotic tRNA intron endonucleases, this enzyme is specific for halophilic tRNA(Trp) substrates. The partially purified enzyme is not capable of removing the intron from a yeast pre-tRNA(Phe) substrate. Analysis of the enzyme's ability to cleave tRNA(Trp) substrates lacking exon sequences demonstrated that the mature tRNA-like structure is not required in the substrate. A substrate retaining the intact intron and only the anticodon stem and loop exon regions was efficiently cleaved. Deletions within the intron indicated that the intron was not a primary site for recognition by the endonuclease; however, its presence affects the efficiency of the cleavage reaction. The possible relationship of this enzyme to other RNA endonucleases is discussed.
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PMID:A tRNA(Trp) intron endonuclease from Halobacterium volcanii. Unique substrate recognition properties. 319 21

In order to determine the mechanism and enzyme(s) responsible for 3' processing of tRNA precursors, we have developed an in vitro processing system that uses as substrates two SP6 RNA polymerase-generated transcripts of the gene for tRNA(Tyrsu3)+ that contain 49 extra 5'-nucleotides and either 5 or 25 extra 3'-nucleotides. A high speed supernatant fraction from an Escherichia coli strain deficient in five ribonucleases was found to accurately process both tRNA precursors in this system to the size of mature tRNA(Tyr). Final 3' end processing of each precursor occurs in an exonucleolytic manner to generate the correct 3' terminus; however, a prior endonucleolytic cleavage also is observed in processing of the longer precursor. The system requires Mg2+ and works optimally at about 50 mM KCl and pH 8-9. Dialysis of the supernatant fraction leads to loss of processing activity but can be restored to normal by the addition of inorganic phosphate or arsenate. Furthermore, nucleoside diphosphates are a product of the processing reaction. These data indicate that 3' processing in RNase-deficient extracts involves a phosphorolytic reaction. On the other hand, phosphate is not required for processing in RNase+ extracts, although it does aid in processing of the longer precursor. The usefulness of this in vitro system for studies of tRNA processing and the identity of the phosphate-requiring enzyme are discussed.
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PMID:3' processing of tRNA precursors in ribonuclease-deficient Escherichia coli. Development and characterization of an in vitro processing system and evidence for a phosphate requirement. 327 67

A recombinant plasmid was constructed with six synthetic DNA oligomers such that the DNA sequence corresponding to yeast tRNA(Phe) is flanked by a T7 promoter and a BstNI restriction site. Runoff transcription of the BstNI-digested plasmid with T7 RNA polymerase gives an unmodified tRNA of the expected sequence having correct 5' and 3' termini. This tRNA(Phe) transcript can be specifically aminoacylated by yeast phenylalanyl-tRNA synthetase and has a Km only 4-fold higher than that of the native yeast tRNA(Phe). The Km is independent of Mg2+ concentration, whereas the Vmax is very dependent on Mg2+ concentration. Comparison of the melting profiles of the native and the unmodified tRNA(Phe) at different Mg2+ concentrations suggests that the unmodified tRNA(Phe) has a less stable tertiary structure. Using one additional DNA oligomer, a mutant plasmid was constructed having a guanosine to thymidine change at position 20 in the tRNA gene. A decrease in Vmax/Km by a factor of 14 for aminoacylation of the mutant tRNA(Phe) transcript is observed.
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PMID:Biochemical and physical characterization of an unmodified yeast phenylalanine transfer RNA transcribed in vitro. 327 87


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