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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 kinetics of the RNA chain initiation reaction carried out by RNA polymerase bound to the initiator region of a DNA template have been analyzed. Initiation proceeds in a two-substrate reaction in which the initial binary complex (enzyme-DNA) is transformed into a ternary complex (enzyme-DNA-RNA) by formation of a dinucleoside tetraphosphate and release of inorganic pyrophosphate. In this reaction RNA polymerase serves as a reactant rather than acting catalytically. The concentration of the reacting binary complex decreases throughout the reaction; hence steady state approximations cannot be used. Kinetic equations for an ordered two-substrate reaction are derived. These are most useful for the special case of reaction in the presence of an inhibitor of initiation, such as rifampicin. Equations for the latter instance are solved exactly with recourse to the steady state approximation. It is found that measurements of the extent of the initiation reaction determined at different inhibitor and substrate concentrations can give information about the initiation reaction analogous to that obtained in standard steady state kinetic analysis. This theory is applied to the experimental study of the initiation reaction carried out by Escherichia coli RNA polymerase. It is found that the inhibitor rifampicin, which blocks the initiation reaciton, acts by binding to the same form of the binary complex as the nucleoside triphosphate substrate (ATP or GTP) which is incorporated into the 5' terminus of nascent RNA molecule. The binding of the 5'-terminal nucleoside triphosphate to the enzyme appears to be rate-limiting for the initiation reaction under standard assay conditions. Initiation appears to follow an ordered reaction mechanism; however, the order of addition of the two substrates is still uncertain.
J Biol Chem 1975 Dec 10
PMID:Kinetic analysis of ribonucleic acid chain initiation by Escherichia coli Ribonucleic acid polymerase bound to DNA. 110 16

1. RNA polymerase from Escherichia coli is selectively and strongly retained by a heparin-substituted agarose and can be eluted therefrom by a neutral buffer containing 0.6 M salt. The method is applicable to relatively crude preparations of the enzyme on a preparative scale giving highly purified RNA polymerase in excellent yield. The enzyme obtained by this procedure shows the highest specific activity so far reported and is pure and enriched in factor sigma as indicated by dodecylsulfate gel electrophoresis. 2. Based on the differential affinity of the subunits of the enzyme for the heparin-carrying gel matrix, a method for separation of alpha, beta' + beta and sigma subunits by application of urea and salt-containing buffers is described. Upon recombination and dialysis with urea-free buffer 40-50% of the enzyme activity is restored.
Eur J Biochem 1975 Dec 01
PMID:Rapid isolation of highly active RNA polymerase from Escherichia coli and its subunits by matrix-bound heparin. 110 37

A soluble extract prepared from T7-infected E. coli is able to initiate DNA synthesis on an exogenous T7 DNA template. We have developed a fractionation procedure to resolve and identify the proteins required for T7 DNA synthesis. By this method we have purified the following T7 replication-related proteins (each greater than 50% pure as judged by sodium dodecyl sulfate gel electrophoresis): T7 DNA-binding protein (27,000 daltons), T7 RNA polymerase (105,000 daltons), T7 DNA polymerase (gene 5-protein, 85,000 daltons, plus host-factor), T7 DNA ligase (40,000 daltons), and T7 DNA-priming protein (65,000 daltons). The T7 DNA-priming protein, synthesized between 7.5 and 15 min following infection, was not detectable if the infecting phage carried an amber mutation in gene 4. Using an in vitro complementation assay which specifically measures the stimulation of DNA synthesis in an extract prepared from T7 gene 4-mutant infected cells, we have purified the DNA-priming protein about 2,000-fold. The purified priming protein preparations are essentially free of endonuclease, exonuclease, DNA ligase and DNA polymerase activity, but they do contain measurable DNA-dependent RNA synthetic acitvity. The enzyme is rapidly inactivated by heating to 46 degrees C and by treatment with N-ethylmalemide. In the presence of T7 DNA-binding protein and all four ribonucleoside triphosphates, the DNA-priming protein enables T7 DNA polymerase to initiate DNA synthesis on intact duplex T7 DNA. Closer studies of its enzymatic function as well as of the possible roles of the other proteins in the T7 replication system will be presented in the accompanying paper.
Mol Gen Genet 1975 Dec 01
PMID:Studies on bacteriophage T7 DNA synthesis in vitro. I. Resolution of the T7 replication system into its components. 110 17

When chromatin prepared from WI-L2 lymphocytes by low salt extraction and shearing is centrifuged on a glycerol gradient, one area of the gradient yields chromatin enriched in template activity for Escherichia coli DNA-dependent RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) as compared to Saccharomyces cerevisiae RNA polymerase II (or B). Another area yields chromatin preferred by the eukaryotic enzyme. Kinetic studies indicate that the differences in activity cannot be explained by differences in affinity of the enzymes for the various templates. The DNA isolated from either fraction has a molecular weight of 8.5 X 106. The "yeast active" fraction seems enriched in proteins. Mixing experiments indicate that the yeast enzyme does not alter the template in such a way as to improve it for the bacterial enzyme.
Proc Natl Acad Sci U S A 1975 Dec
PMID:Separation of lymphocyte chromatin into template-active fractions with specificity for eukaryotic RNA polymerase II or prokaryotic RNA polymerase. 110 5

In order to investigate the symmetry of globin gene transcription, complementary RNA (cRNA) was synthesized using rabbit globin complementary DNA (cDNA) as a template for Escherichia coli DNA-dependent RNA polymerase (RNA nucleotidyltransferase). The cRNA hybridized specifically to its own cDNA template but not to sheep cDNA, rabbit globin mRNA, or poly(dT). Hybridization studies with cRNA demonstrated that RNA sequences transcribed from the DNA strand complementary to the globin gene region (anti-strand) were not present in cellular, total nuclear, or fractionated nuclear RNA from rabbit marrow. Such sequences were detected in RNA transcribed from rabbit marrow chromatin by E. coli or sheep liver RNA polymerases, but amounted to less than 50% of the globin mRNA sequences present in the same transcript. The evidence indicates that globin mRNA transcription is predominantly DNA strand specific.
Proc Natl Acad Sci U S A 1975 Dec
PMID:Strand-selective transcription of globin genes in rabbit erythroid cells and chromatin. 110 6

After T4 bacteriophage infection of E. coli a complex series of events take place in the bacterium, including gross inhibition of host transcription and discrete changes in the classes of the genes of T4 that are transcribed. Accompanying these changes in the pattern of transcription one finds T4-induced changes in the RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase). The effects of modified polymerase on transcription can be advantageously analyzed in a DNA-directed cell-free system for protein synthesis. In this system gene activity is measured indirectly by the amounts and types of proteins sythesized. In the DNA-directed cell-free system this modified polymerase, like normal polymerase, transcribes T4 DNA with a high efficiency but transcribes bacteriophage lambda and host DNA very poorly. Polymerase reconstruction experiments show that modification of the alpha subunit of the RNA polymerase is sufficient for inhibition of host transcription. Host transcription is also inhibited in vitro by T4 DNA. This latter type of inhibition is presumed to involve competition between host DNA and T4 DNA for some factor essential for transcription. The T4-modified polymerase transcribes from T4 DNA many of the same genes as normal unmodified polymerase; it also shows a capability for transcribing certain "non-early" T4 genes which is enhanced in the presence of protein-containing extracts from T4-infected cells.
Proc Natl Acad Sci U S A 1975 Dec
PMID:Effects of bacteriophage T4-induced modification of Escherichia coli RNA polymerase on gene expression in vitro. 110 8

A structural gene for the alpha-subunit of RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) has been identified and mapped between spcA and trkA, near 64 min on the E. coli chromosome. It appears to be coordinately expressed and possibly cotranscribed with the genes for ribosomal proteins S11, S4, and L17.
Proc Natl Acad Sci U S A 1975 Dec
PMID:Identification of a gene for the alpha-subunit of RNA polymerase at the str-spc region of the Escherichia coli chromosome. 110 10

Ribonucleic acid (RNA) synthesis in the sorbitol-dependent, fragile yeast mutant VY1160 (Venkov et al., 1974) is rapidly inhibited by rifampin. The growth of the mutant cells and protein synthesis are more slowly affected by the antibiotic, apparently as secondary phenomena. Lower doses of rifampin (50 to 100 mug/ml) preferentially inhibit ribosomal RNA synthesis in comparison to that of messenger RNA and transfer RNA. Transcription and translation of messenger RNA continues in the presence of low doses of rifampin, as evidenced by the unimpaired induction of alpha-glucosidase. Partially purified RNA polymerase II from this mutant, in contrast to that from the parental strain, is strongly inhibited by low concentrations (1 mug/ml) of rifampin, whereas RNA polymerase I from the two strains is similar in behavior. The mutant may be useful for the study of regulatory mechanisms of transcription in eukaryotes.
Antimicrob Agents Chemother 1975 Dec
PMID:Rifampin susceptibility of ribonucleic acid synthesis in a fragile Saccharomyces cerevisiae mutant. 110 80

A non-histone protein has been isolated from Ehrlich ascites tumor chromatin. The minimum molecular weight of this non-histone protein, estimated by sodium dodecyl sulfate gel electrophoresis and amino acid analysis, is approximately 10 to 11,000. This non-histone protein is acidic, contains 2.7% alkalilabile phosphorus, binds to DNA, and inhibits transcription of DNA in vitro by the homologous RNA polymerase. The per cent inhibition of RNA synthesis is not affected by increasing amounts of RNA polymerase, but is reduced by addition of excess DNA. In the presence of the non-histone protein, incorporation of [gamma-32P]ATP into RNA in the in vitro RNA synthesizing system is inhibited, with no apparent change in the average chain length of the RNA product. Inhibition of RNA synthesis is completely eliminated if the DNA template is allowed to interact with ATP prior to the addition of the non-histone protein. These results indicate that the observed repression of in vitro RNA synthesis is due to the effect of the non-histone protein on the DNA, inhibiting the initiation of RNA chain formation.
J Biol Chem 1975 Dec 10
PMID:Inhibition of transcription in vitro by a non-histone protein isolated from Ehrlich ascites tumor chromatin. 119 69

The limiting factor in RNA synthesis by isolated kidney nuclei is RNA nucleotidyltransferase at high salt concentrations but at low salt concentrations template availability becomes limiting. alpha-Amanitin inhibits 85% of the activity at high salt concentrations but only 20-50% of the activity at low salt concentrations. Exogenous DNA is utilized at low salt concentrations [up to 0-1M (NH4)2SO4] but not at high salt concentrations. The effect of increasing salt concentration is mainly to cause an increase in the length of chains synthesized. Initiation rates are not increased by high salt concentrations. The apparent Km for UTP is 8-10 muM at high salt concentrations, indicating that assays performed at low UTP concentrations are likely to give inaccurate results. The activation energy for the reaction at low salt concentration is less than that for the reaction at high salt concentration. The RNA synthesizing capacity of kidney nuclei is dependent on the method of isolation, and preparation by a modification of the Chauveau method (Chauveau et al. 1956) yields the most active nuclei.
Aust J Biol Sci 1975 Dec
PMID:Characterization of the RNA synthesizing activity of isolated kidney nuclei. 122 83


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