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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)

Electron microscopy was used to study the formation of random complexes between Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) and a promoterless fragment (Mbo I-C) of bacteriophage T7 DNA, and to determine the location of the polymerase molecules bound at 3 degrees to the promoter-containing (Hinf)1100 fragment of the same DNA. The value of the Ka of random binding is about 3 times 10(4)M-1 when the enzyme is slowly diluted from its storage condition and is incubated with DNA for up to 2 min at 37 degrees. If dilution is rapid and occurs in a single step, or if incubation extends beyond 5 min, a substantial portion of RNA polymerase is converted to a form that binds randomly with a much greater affinity (about 10(8)M-1). Hence true random binding by RNA polymerase holoenzyme is much weaker than previously thought. However, great caution is required in assessing the extent of random binding where damage to the enzyme may occur. When RNApolymerase holoenzyme is incubated at 0 degrees with promoter-containing fragment (Hinf)1100, complexes form at the same promoter sites utilized at 37 degrees, although the highly stable "open" promoter complex is not formed under these conditions. However, the extent of binding is reduced as compared to promoter complexes formed at 37 degrees. This gives direct evidence for formation of complexes with promoter sites that have properties of the hypothetical "closed"complexes formed between RNA polymerase and duplex DNA.
Proc Natl Acad Sci U S A 1977 Sep
PMID:Electron microscope studies of transient complexes formed between Escherichia coli RNA polymerase holoenzyme and T7 DNA. 33 45

Recombinant DNA molecule of phage lambda formed in Escherichia coli in the presence of chloramphenicol and/or rifampin can be assayed by their biological activity. recA- cells were found to be capable of forming recombinant lambda phage DNA in the presence of chloramphenicol. The relatively high recA-independent recombination observed in this system contrasts with the relatively low recA-independent recombination when recombinant phage particles rather than recombinant DNA are titrated. Formation of the recombinant DNA was suppressed by the the addition of rifampin. The introduction of the rif-r mutation into host bacteria made their recombination activity rifampin-resistant. These results show that DNA-dependent RNA polymerase (EC 2.7.7.6) is involved in this recA-independent pathway of recombination, which is named the "Rpo pathway." This is distinct from Red, Int, RecBC, RecE, or Der pathways of recombination. Crossover was much more frequent in the N-PL-cI and cI-PR-O regions than in the A-D and O-S regions. The crossover seems to occur in the regions that are transcribed actively. Some local change of DNA structure caused by transcription might be required for the Rpo pathway of recombination.
Proc Natl Acad Sci U S A 1977 Sep
PMID:Involvement of DNA-dependent RNA polymerase in a recA-independent pathway of genetic recombination in Escheria coli. 33 50

The complexes of T2 DNA with RNA polymerase of Escherichia coli were studied by two methods: kinetic formaldehyde method with preliminary fixation of complexes with low formaldehyde concentrations, and electron microscopy. For electron-microscopic investigations the effect of different conditions of formaldehyde fixation for DNA-RNA-polymerase complexes was studied and optimal fixation conditions were found. The suggested fixation method for DNA-RNA-polymerase complexes allows investigation of RNA polymerase molecule distribution on DNA in a wide range of conditions (ionic strength of the solution, weight ration of enzyme to DNA etc.). The comparison of the concentration of RNA polymerase molecules bound to DNA, determined by electron microscopy, and the concentration of defects in DNA as determined by the kinetic formaldehyde method, showed their coincidence. The electron-microscopic procedure was used to make maps of RNA polymerase distribution on T7 DNA. A correlation between the binding regions of the enzyme and the genetic map of early DNA T7 region was found.
Eur J Biochem 1977 Sep 15
PMID:Investigation of the binding of Escherichia coli RNA polymerase to DNA from bacteriophages T2 and T7 by kinetic formaldehyde method and electron microscopy. 33 47

The cell-free transcription of the argF and argI genes of the arginine biosynthetic regions is described using an S-30 system capable of coupled in vitro transcription-translation. Template DNA isolated from two independently isolated arginine transducing phages was used in this work. Steady state mRNA synthesis was observed which was attributed to RNAase degradation. Regulation of argF mRNA synthesis, directed by the argF gene carried on the specialized transducing phage phi80dargF is effected to the extent of at least 95% by the arginine holorepressor at the transcriptional stage and at least 80% of the regulation of the expression of the argI gene is mediated at the transcriptional stage. Evidence is presented which indicates that the arginine holorepressor prevents RNA polymerase from binding to the arginine promoter and suggests that the operator and promoter sites may overlap.
Mol Gen Genet 1977 Sep 21
PMID:Transcription of the argF and argI genes of the arginine biosynthetic regulon of Escherichia coli K12, performed in vitro. 33 19

Escherichia coli RNA polymerase has been shown to bind to a limited number of Hind and Hae III restriction enzyme fragments. On S13 replicative form DNA there are three major binding sites, and the locations correlate with promoter sites at the beginning of genes a and B and a site overlapping gene D and the beginning of gene E. Two less definite binding sites have been localized, one in gene F and one at the gene G-H junction. In phiX174 replicative form DNA, five sites, each with apparently similar binding properties, have been located, four of which correspond exactly to binding sites in S13. One site, at the beginning of the B gene, could not be assigned to exactly the same location found in S13. This was due in part to differences in the restriction cleavage maps in the area of the DNA and possibly to the higher background of nonspecific binding in the phiX174 experiments. The location of two of the phiX174 sites at the beginning of genes A and D-E corresponds very well with transcription data, but the site at the start of the B gene indicates the promoter site is closer to the initiation sequence of the B protein than was previously suggested on the basis of transcription data.
J Virol 1978 Sep
PMID:Localization of Escherichia coli RNA polymerase binding sites on bacteriophage S13 and phiX174 DNA: alignment with restriction enzyme maps. 35 30

The interaction between Escherichia coli RNA polymerase and a restriction fragment of coliphage T7 DNA containing four promoter sites for the coli enzyme has been studied by difference uv absorption spectroscopy in a low ionic strength buffer containing 10 mm MgCl2 and 50 mM KCl. The binding of the enzyme to the DNA is accompanied by a hyperchromic shift which shows a maximum around 260 nm, and increases with increasing temperature in the temperature range studied (4-40 degrees C). Measurements were also carried out with whole T7 DNA and a restriction fragment containing no promoter site. A comparison of the results obtained with the various DNAs suggests that the binding of an RNA polymerase to a promoter site in the low ionic strength medium causes the disruption of a short segment of the DNA helix, of the order of ten pairs; the binding of an enzyme molecule to a promotor site appears to have a cooperative effect on the binding of the enzyme molecules to adjacent non-promoter sites with concomitant disruption of DNA base pairs.
Nucleic Acids Res 1978 Sep
PMID:Physiochemical studies on interactions between DNA and RNA polymerase. Ultraviolet absorption measurements. 36 Jan 69

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.
Mol Gen Genet 1978 Sep 20
PMID:RNA polymerase mutant with altered sigma factor in Escherichia coli. 36 59

Upon addition of excess one carbon metabolites (including serine)bacteria stop growing because of isoleucine starvation. After such treatment stringent bacteria rapidly resume normal growth whereas relaxed mutants remain unable for some time to grow. We show here that this is due to a lack of derepressibility of ilv genes after the starvation period. Results are also presented which show that RNA polymerase structural mutants may be selected among the clones resistant to a mixture of serine, methionine and glycine, in relA- strains. Finally circumstancial evidence suggests that the one carbon metabolism may be involved in a process controlling isoleucine metabolism.
Mol Gen Genet 1978 Sep 20
PMID:Correlation between the serine sensitivity and the derepressibility of the ilv genes in Escherichia coli relA- mutants. 36 63

The antibiotic rifampicin inhibits transcription initiation, but not the elongation and completion of nascent RNA transcripts. Addition of low concentrations of rifampicin only partially blocks initiation but at the same time specifically alters the general pattern of transcription in the culture. The transcription of genes specifying the beta and beta' subunits of RNA polymerase, and to a lesser extent of the genes specifying the RNA and protein components of the ribosome, was specifically stimulated relative to total transcription. In contrast, the transcription of the lactose operon was selectively reduced. These results are consistent with the ideas that the level of expression of the genes specifying the beta and beta' subunits is sensitive to the general rate of RNA synthesis in the culture, and that the expression of the beta and beta' RNA polymerase genes is related to the expression of ribosome component genes.
Mol Gen Genet 1978 Sep 20
PMID:Gene expression in Escherichia coli B/r during partial rifampicin-mediated restrictions of transcription initiation. 36 68

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.
Chem Biol Interact 1979 Sep
PMID:Non-random nature of 2-acetylaminofluorene-induced alterations of DNA template capacity. 38 7


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