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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)

By treatment with tRNA in the presence of 1 mM MgCl2, a chromatin preparation was obtained containing all five major histone fractions but lacking a considerable portion of non-histone proteins. This chromatin preparation as well as chromatin extracted with 0.6 M NaCl (depleted of H1 histone and some non-histone proteins) were characterized in respect of solubility and chromatin DNA accessibility. Both samples possessed practically the same solubility in the presence of 0.15 M NaCl and 1 mM MgCl2. The solubility of tRNA-treated chromatin in 5 and 10 mM MgCl2 was higher than that of salt-extracted chromation. The accessibility of the DNA of these chromatin preparations was tested with DNA-dependent RNA polymerase of Escherichia coli as a probe, using procedure that permits measurement of binding site frequency. Both tRNA-treated and salt-extracted chromatin contained as many as 33% and untreated chromatin as few as 4% of the number of binding sites found on protein-free DNA. These results demonstrate that at least in part the non-histone proteins are responsible for salt-induced insolubility and low DNA accessibility of chromatin, thus revealing the importance of non-histone proteins in the maintenance of an overall chromatin structure.
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PMID:Changes in chromatin properties after partial extraction of non-histone proteins. 34 51

We have investigated the nonspecific interactions of Escherichia coli RNA polymerase core and holoenzyme with double-stranded (ds) and single-stranded (ss) DNA. Binding constants for these interactions as functions of such solution variables as monovalent and/or divalent cation concentration, temperature, or pH were determined by the method of deHaseth et a. [deHaseth, P.L., Gross, C.A., Burgess, R.R. and Record, M.T. (1977), Biochemistry 16, 4777--4783] from analysis of the elution of the proteins from small columns containing immobilized DNA. This technique, although as yet empirical, has been demonstrated to yield accurate binding constants fot the nonspecific interation of lac repressor with ds DNA. We find that observed binding constants (Kobsd) are extraordinarily sensitive functions of the monovalent cation concentration for the interactions of both core and holoenzyme with ds DNA. In the absence of divalent cations, the derivatives --(d log Kobsd/d log [Na+]) are 11 +/- 2 for the holo--ds DNA interaction and 21 +/- 3 for the core--ds DNA interaction. Consequently, approximately 11 and 21 low-molecular-weight ions are released, iin the thermodynamic sense, in the formation of the holo--ds and core--ds complexes, respectively (Record, M.T., Jr., Lohman, T.M., and deHaseth, P.L. (1976), J. Mol. Biol. 107, 145--158; Record, M.T., Jr., Anderson, C.F., and Lohman, T.M. (1978), Q. Rev. Biophys., in press). Ion release is a thermodynamic driving force for these nonspecific interactions and causes the stability of the complexes to increase very substantially with a reduction in monovalent ion concnetration. Possible molecular models which account for the different salt sensitivities of the holo--ds and core--ds complexes are discussed. Effects of the competitive ligand Mg2+ on these interactions are also examined. Substantial ion release (approximately 18 monovalent ions) also accompanies the interaction of either holo or core polymerase with ss DNA. Over the range of ion concentrations investigated the holo--ss interaction is substantially stronger than the core--ss interaction; furthermore, we conclude that the interactions of polymerase with ss DNA are, in general, stronger than the nonspecific interations of the enzyme with ds DNA. It is likely that the nonspecific interactions of RNA polymerase with DNA have physiological relevance. Not only is it plausible to assume that the same regions of the protein are involved in both specific and nonspecific interactions, but in addition nonspecific interactions of RNA polymerase and DNA may play role in determining the availability of this protein, in both the thermodynamic and the kinetic sense, for promoter binding and RNA chain initiation [von Hippel. P.H., Revzin, A., Gross, C.A., and Wang, A.C. (1974), Proc. Natl. Acad. Sci U.S.A. 71, 4808--4812]. Consequently, the strong dependences of the nonspecific interactions of RNA polymerase on ionic conditions suggest the possibility of a modulating role of ion concentrations in the control of transcription.
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PMID:Nonspecific interactions of Escherichia coli RNA polymerase with native and denatured DNA: differences in the binding behavior of core and holoenzyme. 35 Feb 71

A structural gene for sigma factor (rpoD) of DNA-dependent RNA polymerase (RNA nucleotidyltransferase; nucleoside-triphosphate: RNA nucleotidyltransferase, E.C. 2.7.7.6) was mapped precisely by a set of F' factors including those already published (Proc. Natl. Acad. Sci. USA. 74, 1831-1835 (1977)). Based on the result that rpoD is located at the dnaG-uxaAC region, a number of mutants containing a temperature-sensitive mutation at or near the uxaA gene were isolated by localized mutagenesis. One of these mutants was found to produce RNA polymerase altered in both thermostability and optimum salt concentration as a result of structural alteration of sigma factor. This mutation, U303, maps at 66 min on the genetic map of E. coli, near the dnaG locus, and affects normal growth of cells.
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PMID:RNA polymerase mutant with altered sigma factor in Escherichia coli. 36 59

Purified core histones (H2A, H2B, H3, and H4) and bacteriophage T7 DNA have been reconstituted to form a nucleoprotein complex, and the properties of this complex as a template for transcription by Escherichia coli RNA polymerase have been studied. At low ionic strength, RNA chain elongation rates are slow, and the chains produced even after long incubation are short. At higher salt concentrations, chain-elongation rates approach those on naked DNA. Since the salt concentrations used are not in themselves sufficient to dissociate the histones from the DNA, some mechanism must exist that permits passage of the polymerase through histone-covered regions.
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PMID:Transcription of histone-covered T7 DNA by Escherichia coli RNA polymerase. 36 33

In vivo, termination of transcription at the attenuator site of the tryptophan (trp) operon of E. coli is influenced by the protein termination factor rho. In vitro, termination does not depend on rho factor, and is very efficient in a purified system consisting only of RNA polymerase, the DNA template, nucleoside triphosphates, and buffer. The extent of termination in this system is unaffected over a wide range of salt and nucleoside triphosphate concentration. However, there is a 10-fold stimulation of trp leader mRNA synthesis if rho factor is present during the transcription reaction. This stimulation occurs only at low molar ratios of polymerase to template, and can be blocked by rifampicin. It is thus most likely due to the recycling of RNA polymerase molecules that have been released from the attenuator site by rho factor. In fact, transcription of the trp leader region in vitro results in the fomration of a stable termination complex which can be observed on sucrose gradients or by binding to nitrocellulose filters. These data indicate that a major function of rho at the trp attenuator is to release completed transcripts from a pre-formed termination complex, rather than to cause the cessation of elongation.
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PMID:The attenuator of the tryptophan operon in E.coli: rho-mediated release of RNA polymerase from a transcription termination complex in vitro. 37 Jul 76

The coenzyme A-glutathione mixed disulfide (CoASSG), when complexed with iron, is capable of inhibiting the RNA polymerase of Escherichia coli. A modified procedure involving a short time of exposure to high salt allowed the reliable preparation of CoASSG-Fe which was active in inhibiting RNA polymerase. The CoASSG-Fe complex acted as a noncompetitive inhibitor for the incorporation of all four nucleoside triphosphates but had a greater effect on GMP and CMP incorporation than AMP and UMP incorporation. Neither temperature nor ionic-strength changes affected CoASSG-Fe inhibition, and the use of rifampicin showed that CoASSG-Fe did not inhibit either the initiation or elongation processes of the polymerase. CoASSG-Fe was a more effective inhibitor at low DNA-template concentrations and it was more effective in inhibiting the incorporation of CMP and GMP on simple dG-dC containing templates and the asymmetric polymer poly d(T-C) . poly d(G-A). The inhibition of transcription of poly d(I-C) was less effective than the inhibition of transcription of poly d(G-C). Equilibrium dialysis in microdialysis cells showed that CoASSG-Fe could associate with DNA in the absence of RNA polymerase.
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PMID:Partial characterization of the mode of inhibition of Escherichia coli RNA polymerase by the mixed disulfide, CoASSG. 37 69

The DNA-dependent RNA polymerase I (or A) from the lower eukaryote Aspergillus nidulans has been purified on a large scale to apparent homogeneity by homogenizing the fungal hyphae in liquid nitrogen, extraction of the enzyme at high salt concentration, precipitation of RNA polymerase activity with polymin P (a polyethylene imine), elution of the RNA polymerase from the polymin P precipitate, ammonium sulphate precipitation, molecular sieving on Bio-Gel A-1.5m, binding to ion-exchangers and DNA-cellulose affinity chromatography. By this procedure 1.6 mg of RNA polymerase I can be purified over 2000-fold from 500 g wet weight of starting material with a yield of 30--35%. The isolated RNA polymerase I is stable for several months at -20 degrees C. The subunit compostion has been resolved by polyacrylamide gel electrophoresis on two-dimensional gels, using either non-denaturing of 8 M urea (pH 8.7) cylindrical gels in the first dimension and sodium dodecyl sulphate slab gels in the second dimension. The putative subunits have molecular weights of 190,000, 135,000, 63,000, 62,000, 43,000, 29,000, (28,000), 16,000 and probably 13,000 and 12,000. Two distinct forms of RNA polymerase I (Ia and Ib) have been resolved by DEAE-Sephadex A-25 chromatography showing ample differences in enzymatic properties and subunit pattern. Additional information is given on RNA polymerase II (or B) which appears to be highly insensitive to alpha-amanitin at concentrations up to 400 micrograms/ml.
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PMID:RNA polymerase from the fungus, Aspergillus nidulans. Large-scale purification of DNA-dependent RNA polymerase I (or A). 38 Sep 97

The fixation of tRNA to Escherichia coli RNA polymerase has been investigated. Bound and free tRNA have been separated and quantified after filtration through cellulose nitrate filters, centrifugation or sucrose gradients or electrophoresis in polyacrylamide gels. We detect no differences between the fixation of E. coli fMet-tRNAfMet, Met-tRNAmMet or uncharged unfractionated tRNA to RNA polymerase. Tight complexes, with a long residence time, are formed between core enzyme and tRNA with a dissociation constant of less than 1 nM. Complexes exist between tRNA and both monomer and dimer forms of the core enzyme. In the monomer complex, one tRNA is bound per alpha 2 beta beta' unit, whereas in the dimer complex only 0.5 tRNA molecule is fixed per alpha 2 beta beta' unit. In contrast to the core enzyme, very little tRNA fixes tightly to the holoenzyme at salt concentrations greater than 80 mM. At lower salt concentrations tRNA fixation results in a loss of sigma subunit from the holo enzyme to the resulting core enzyme where it binds tightly. DNA fixation reduces the binding of tRNA to RNA polymerase and tRNA fixation reduces the binding of DNA. However, binding of DNA to polymerase is not competitive with binding of tRNA, and ternary complexes between RNA polymerase, DNA and tRNA are shown to exist. Our results are discussed in relation to other studies concerning the effects of tRNA upon RNA polymerase.
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PMID:On the binding of tRNA to Escherichia coli RNA polymerase. 38 19

Two ribonuclease H activities have been found in yeast RNA polymerase A. The nuclease activities comigrated with subunits A49 (Mr = 49,000) and A40 (Mr = 40,000), after electrophoresis in a sodium dodecyl sulfate polyacrylamide gel containing [32P](rG)n . (dC)n as substrate. Both activities were also found, among other nucleases, in a high salt chromatin extract. Several lines of evidence suggest that the chromatin RNase H of 49,000 daltons (RNase H49) is the same protein as subunit A49. They co-migrate on sodium dodecyl sulfate-gel electrophoresis, have the same chromatographic properties, and dissociate simultaneously from RNA polymerase A. Fractions containing RNase H49 stimulate RNA synthesis by RNA polymerase A* lacking A49 and A34.5 subunits. Finally, limited proteolysis of the protein band having RNase H49 activity yields the characteristic fingerprint of the A49 subunit. This subunit, therefore, exists in two states: bound to chromatin and associated with RNA polymerase A. On the other hand, it is not yet clear whether the RNase H activity of 40,000 daltons, associated with RNA polymerase A, is due to the A40 subunit or whether it represents a trace contamination by a very active nuclease tightly bound to the enzyme.
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PMID:Identification of two different RNase H activities associated with yeast RNA polymerase A. 38 60

The interaction between RNA polymerase and the E. coli ribosomal (r) RNA promoter(s) of the rrnE operon has been studied by the filter-binding method. The extent of complex formation between RNA polymerase and rrnE promoter(s) is salt-dependent; ppGpp specifically inhibits interaction of RNA polymerase with the rrnE promoter(s). A tentative model is proposed for the molecular events in the early steps of rRNA initiation: a transition of the primarily formed, labile RNA polymerase-rRNA promoter complex to a more stable form is the determining step. This step is salt-sensitive; ppGpp acts on this "isomerization".
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PMID:Interaction between RNA polymerase and a ribosomal RNA promoter of E. coli. 38 48


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