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 L and NS proteins of vesicular stomatitis virions (New Jersey serotype) were solubilized with Triton X-100 and high-salt buffer and recombined with purified nucleocapsids under conditions similar to those used to reconstitute transcriptase activity in vitro. The nucleocapsid-bound L and NS proteins were separated from unbound proteins on a glycerol gradient. The rebinding of L and NS proteins mimics the in vivo binding in that at saturation the ratio of L and NS molecules to N molecules is approximately the same as observed in the intact virion. L and NS proteins were separated and added back independently and in combination to the template. The purified NS protein bound to the template in the absence of L protein. However, the L protein binding appeared to depend on the presence of NS protein. The presence of Mg2+ and nucleotides, which is required for transcription, was not necessary for the rebinding of L and NS proteins.
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PMID:Rebinding of transcriptase components (L and NS proteins) to the nucleocapsid template of vesicular stomatitis virus. 21 81

Three temperature-sensitive mutant strains for RNA polymerase beta or beta' subunits (carrying mutations tsx, A2R7 and R120) were used in order to investigate the dependence of the induced lac expression on stimulation by cyclic AMP after the shift to non-permissive temperature. High temperature lowered the rate of beta-galactosidase synthesis. However, the low rate of synthesis could be strongly increased by cyclic AMP (30, 2.4 and 5.7-fold increases for tsX, A2R7 and R120 mutants, respectively). At the permissive temperature stimulation by cyclic AMP was less than 1.4-fold (minimal medium supplemented with glycerol). The results suggest that the maximal expression of the lac operon is saturated, that is, a hypothetical increase in RNA polymerase or cAMP-CRP concentration in the cell with not enhance the expression. The concept of saturation explains why it was possible to increase the beta-galactosidase synthesis in conditions of limited promoter binding activity of RNA polymerase through increase in concentration of cyclic AMP-CRP complex in the cell (addition of cyclic AMP) to the values higher than that observed on glycerol.
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PMID:Expression of the lac operon in RNA polymerase mutants of Escherichia coli K12. 22 41

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...
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PMID:On the DNA polymerase III of mouse myeloma: partial purification and characterization. 23 42

The synthesis of ribosomal ribonucleic acid (rRNA), transfer RNA (tRNA) and messenger RNA (mRNA) was measured in Escherichia coli B/r after the addition of 100 mug of chloramphenicol (CAM) per ml to cultures growing either in one of three minimal media (succinate, glycerol, or glucose) or in one of the same three media supplemented with 20 amino acids. (i) During CAM treatment, rRNA and tRNA were synthesized in the same relative proportions (85:15) as during exponential growth. The faster accumulation of tRNA relative to rRNA in CAM was due to a decreased stability of rRNA that is synthesized in the presence of or immediately before the addition of CAM. (ii) CAM stimulated the synthesis of rRNA and tRNA two- to eightfold. The results fell into two groups; one group was from studies done in minimal media and the other was from amino acid-supplemented media. In each group the stimulation decreased with increasing growth rate of the culture during exponential growth before the addition of CAM; however, the stimulation in minimal media was lower than that in amino acid-supplemented media. (iii) CAM caused an increase in the proportion of rRNA and tRNA synthesis and a corresponding decrease in the proportion of mRNA synthesis. In minimal media, the residual proportion of mRNA synthesis after CAM treatment was 10 to 15% of total RNA synthesis; in amino acid-supplemented media this proportion was 0 to 10%. In either case, the residual proportion of mRNA synthesis was independent of the proportions observed during exponential growth in these media. (iv) The absolute rate of mRNA synthesis decreased severalfold with the addition of CAM; i.e., the rate of synthesis of rRNA and tRNA was increased at the expense of mRNA synthesis. (v) During exponential growth, the fraction of the instantaneous rate of total RNA synthesis that corresponds to mRNA is a function of both the growth rate and the presence or absence of amino acids in the growth medium: in the absence of amino acids, this fraction decreased with increasing growth rate; in the presence of amino acids, the fraction increased slightly with growth rate. These results are consistent with a regulation of rRNA and tRNA synthesis at the transcriptional level, e.g., with a CAM-induced increase in the affinity of RNA polymerase for the rRNA and tRNA promoters. The results also suggest the occurrence of a regulation of RNA polymerase enzyme activity, i.e., of an activation of RNA polymerase that is inactive during exponential growth. A distinction between these alternatives requires measurements of the rRNA chain growth rates during CAM treatment.
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PMID:Chloramphenicol-induced changes in the synthesis of ribosomal, transfer, and messenger ribonucleic acids in Escherichia coli B/r. 32 74

S. tyrphimurium strain BY324 is temperature sensitive due to a mutation (rpo C32) in the gene for the RNA polymerase beta' subunit. Transcription of T7 DNA by RNA polymerase purified from this strain is temperature sensitive in vitro. The enzyme is slightly defective in template binding and RNA chain initiation, but the major defect is in RNA chain elongation. The rate of RNA chain elongation is reduced 4-5 fold relative to wild-type. RNA chain termination does not appear to be affected by the beta' mutation. While the elongation defect is suppressed by glycerol or dimethylsulfoxide, the initiation defect is not. Possible roles for the beta' subunit in enzyme function are discussed in light of these results.
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PMID:Multiple effects of an RNA polymerase beta' mutation on in vitro transcription. 33 27

The "host shutoff" function of bacteriophage T7 involves an inactivation of the host Escherichia coli RNA polymerase by an inhibitor protein bound to the enzyme. When this inhibitor protein, termed I protein, was removed from the inactive RNA polymerase complex prepared from T7-infected cells by glycerol gradient centrifugation in the presence of 1 M KCl, the enzyme recovered its activity equivalent to about 70 to 80% of the activity of the enzyme from uninfected cells. Analysis of the activity of E. coli RNA polymerase from E. coli cells infected with various T7 mutant phages indicated that the T7 gene 2 codes for the inhibitor I protein. The activity of E. coli RNA polymerase from gene 2 mutant phage-infected cells, which was about 70% of that from uninfected cells, did not increase after glycerol gradient centrifugation in the presence of 1 M KCl, indicating that the salt-removable inhibitor was not present with the enzyme. It was found that the reduction in E. coli RNA polymerase activity in cells infected with T7(+) or gene 2 mutant phage, i.e., about 70% of the activity of the enzyme compared to that from uninfected cells after glycerol gradient centrifugation in the presence of 1 M KCl, results from the function of T7 gene 0.7. E. coli RNA polymerase from gene 0.7 mutant phage-infected cells was inactive but recovered a full activity equivalent to that from uninfected cells after removal of the inhibitor I protein with 1 M KCl. E. coli RNA polymerase from the cells infected with newly constructed mutant phages having mutations in both gene 2 and gene 0.7 retained the full activity equivalent to that from uninfected cells with or without treatment of the enzyme with 1 M KCl. From these results, we conclude that both gene 2 and gene 0.7 of T7 are involved in accomplishing complete shutoff of the host E. coli RNA polymerase activity in T7 infection.
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PMID:"Host shutoff" function of bacteriophage T7: involvement of T7 gene 2 and gene 0.7 in the inactivation of Escherichia coli RNA polymerase. 33 32

Bacteriophage T7-coded inhibitor of Escherichia coli RNA polymerase, termed I protein, was purified from an inactive E. coli RNA polymerase-I protein complex isolated from phage T7-infected cells. A molecular weight of about 7,000 to 9,000 was assigned to the purified I protein by acrylamide-sodium dodecyl sulfate gel electrophoresis, Sephadex G-50 gel filtration, and glycerol gradient centrifugation analysis. I protein inhibits initiation of RNA synthesis by directly binding to the RNA polymerase holoenzyme and prevents the binding of the enzyme to the promoter sites on the template T7 DNA. However, once a highly stable transcriptional preinitiation complex between RNA polymerase holoenzyme and T7 DNA is formed at the promoter site on T7 DNA in the absence of nucleoside triphosphates, I protein does not inhibit the initiation of RNA synthesis by this preformed complex upon addition of nucleoside triphosphates. RNA synthesis by the core RNA polymerase and the binding of core RNA polymerase with template DNA are not inhibited by I protein, although a partial association between the core enzyme and I protein can be observed. I protein does not bind to sigma factor or T7 DNA. Therefore, binding of I protein with the RNA polymerase, which results in the inhibition of initiation of RNA synthesis, requires the presence of sigma factor in the RNA polymerase holoenzyme form.
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PMID:I protein: bacteriophage T7-coded inhibitor of Escherichia coli RNA polymerase. 33 33

Transcription of tRNA genes carried by transducing bacteriophages phi80psu3+ (tRNA1Tyr) and lambdah80T (tRNA2Tyr, tRNA2Glysu36+, tRNA3Thr) was studied in vitro in a system consisting of whole bacteriophage DNA and purified RNA polymerase. In contrast to unusual requirements for tRNA1Tyr gene transcription from DNA fragments, the transcription on whole bacteriophage DNA was found to be relatively not salt sensitive, did not require glycerol and rifampicin-resistant complexes with RNA polymerase were formed in the absence of nucleoside triphosphates. Termination factor rho stimulated the transcription of the tRNA genes as well as that of 4S RNA on lambdah80T DNA template. The stimulatory effect of rho was abolished by rifampicin and seems to be due to the release of RNA polymerase and reinitiation of transcription.
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PMID:In vitro transcription of E. coli tRNA genes. 33 3

Yeast nuclear RNA polymerase III was purified by batch adsorption to phosphocellulose, followed by ion-exchange chromatography on DEAE-Sephadex and affinity chromatography on DNA-Sepharose. Polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band which contained polymerase activity. The molecular weight estimated by sedimentation velocity centrifugation in a glycerol gradient was 380 000. Enzyme activity was inhibited 50% at 0.1 mM 1,10-phenanthroline and 100% of 1.0 mM, but was restored when 1,10-phenanthroline was removed by dialysis. Enzyme activity was not inhibited by 7,8-benzoquinoline, a nonchelating structural analogue of 1,10-phenanthroline. These results strongly suggest that inhibition of enzyme activity occurs by the formation of a reversible enzyme-zinc-phenanthroline ternary complex. The zinc content, measured by atomic absorption spectroscopy, was 2 g-atoms per mol of enzyme. Zinc was not removed from the enzyme by gel filtration on Sephadex G-25, by passage through Chelex-100 resin, or by dialysis against buffer containing 1,10-phenanthroline. Enzyme-bound zinc was removed by dialysis after denaturation of the enzyme with heat and sodium dodecyl sulfate. Enzyme-bound zinc did not exchange with free zinc. These results establish yeast nuclear RNA polymerase III as a zinc metalloenzyme.
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PMID:Saccharomyces cerevisiae DNA-dependent RNA polymerase III: a zinc metalloenzyme. 33 47

Male rats were fed a diet containing 0.03% (w/w) 2-acetylaminofluorene (AAF) and their hepatic DNA was isolated and transcribed with E. coli RNA polymerase. Ingestion of the carcinogen-containing diet for 4 days substantially reduced the template capacity of the isolated DNA. This reduction in template capacity was due to an apparent decreased RNA chain size (up to 50%), with no significant changes in initiation or re-initiation of RNA synthesis. This premature termination of RNA synthesis was accompanied, in some instances, by a reduced rate of RNA chain elongation. When the rats were returned to a basal diet for 7 days following 4 days of AAF ingestion, template capacity and RNA chain size returned to control values. Fractionation of hepatic chromatin on a glycerol gradient revealed that inhibition of DNA template capacity occurs on portions exhibiting characteristics of expressed, as well as those with characteristics of repressed, segments of the genome. In contrast, the DNA isolated from a small, highly condensed chromatin fraction (15% of total chromatin-DNA) showed no significant reduction in total template capacity. Analysis of the fidelity of RNA synthesis on this DNA template was performed by determining the rate of addition of individual nucleotide triphosphates to a growing RNA chain. Large reductions in the rates of adenosine and uridine polymerization were observed while no changes in guanosine or cytidine polymerization were found. This suggests the presence of functionally significant carcinogen-induced modifications of adenine. The inhibition in the rate of adenosine and uridine polymerization was reversed when the animals were placed on a basal diet after AAF ingestion.
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PMID:Non-random nature of 2-acetylaminofluorene-induced alterations of DNA template capacity. 38 7


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