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)

Escherichia coli RNA polymerase contacts promoter DNA at two regions (the -10 and -35 regions) which are separated by a segment of spacer DNA. Previously we showed that base substitutions in the spacer DNA can affect promoter strength both in vitro and in vivo; these results were interpreted to reflect altered structural properties of the substituted DNAs. Here we provide experimental support for this interpretation. The pattern of cleavage of the promoters with Neurospora crassa endonuclease and the reactivity of their guanine residues with dimethyl sulfate (DMS) suggest that the structures of the spacer DNAs in the promoters with altered transcriptional activities are distinct. In addition, the binding of RNA polymerase to the latter promoters induces characteristic enhancements in the extent to which specific guanine residues in the spacer DNAs react with DMS. We propose that for these promoters the substitutions in the spacer DNAs have affected the relative orientation of the -10 and -35 regions. The observed differences in promoter activity then would reflect the requirement for realignment of these regions during the process of open complex formation; we postulate that two such realignments occur.
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PMID:Promoter recognition by Escherichia coli RNA polymerase. Influence of DNA structure in the spacer separating the -10 and -35 regions. 305 Jan 26

The effects of an inhibitory monoclonal antibody (mAb) raised against the beta subunit of the Escherichia coli RNA polymerase were determined on the kinetics and structural interactions during formation of the open promoter complex (RPo). Analysis of the kinetics of abortive initiation on linear and supercoiled templates of the lac and TAC16 promoters showed that abortive synthesis by mAb 210E8-RNA polymerase varied as a function of DNA topology. A kinetic analysis of RPl formation on the supercoiled lac UV5 promoter showed that mAb 210E8 effected a slight alteration in the isomerization rate and no effect on the initial rate of RNA polymerase binding to the promoter. The potent inhibition of initiation with linear promoters by mAb 210E8 was not apparent when the promoters were assayed in their supercoiled forms. Abortive synthesis with the TAC16 promoter was accompanied by an mAb 210E8 induced hindrance of ApUpU but not UpGpU synthesis. The data indicate that the inhibition by mAb 210E8 with the supercoiled TAC16 promoter is further alleviated when the spacer length is shifted from 16 base pairs (ApUpU formation) to 18 base pairs (UpGpU formation). When DNase I and dimethyl sulfate were used to probe DNA structure, mAb 210E8 was found to alter polymerase interactions with the lac promoter. DNase I footprinting indicated that the structural interactions for lac P+ promoter-RNA polymerase complexes were slightly altered in the presence of mAb 210E8. Treatment of the RNA polymerase-lac UV5 complex with dimethyl sulfate revealed an alternate mode of RNA polymerase interaction with essential guanine contacts which was intermediate between a fully protected and free promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effects of an anti-beta monoclonal antibody on the interaction of the Escherichia coli RNA polymerase with the lac and TAC promoters. 329 50

The rhaC gene, whose product is the positive activator of the genes required for L-rhamnose utilization, has been cloned along with the rhamnose structural genes. The rhaC sequence shows two partially overlapping reading frames, encoding two proteins of molecular weight 32,000 and 35,000 RhaS and RhaR. Both proteins show significant homology to AraC, the positive activator of the arabinose operon. S1 mapping located transcriptional start points and showed that RhaR, and possibly RhaS, positively regulate transcription from the structural gene promoters as well as transcription from their own promoter. In-vivo dimethyl sulfate footprinting and DNase I footprinting indicate that the RhaR protein may bind to DNA elements upstream from its RNA polymerase binding site.
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PMID:Positive regulation of the Escherichia coli L-rhamnose operon is mediated by the products of tandemly repeated regulatory genes. 331 63

Two sets of experiments have been performed to test the DNA loop model of repression of the araBAD operon of Escherichia coli. First, dimethyl sulfate methylation protection measurements on normally growing cells show that the AraC regulatory protein occupies the araI site in the presence and absence of the inducer arabinose. Similarly, the araO2 site is shown to be occupied by AraC protein in the presence and absence of arabinose; however, its occupancy by AraC is greatly reduced when araI and adjacent sequences are deleted. Thus, AraC protein binds to araO2 cooperatively with some other component of the ara system located at least 60 base pairs away. Second, the mutational analysis presented here shows that the DNA components required for repression of araBAD are araI, araO2, and perhaps the araBAD operon RNA polymerase binding site.
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PMID:The DNA loop model for ara repression: AraC protein occupies the proposed loop sites in vivo and repression-negative mutations lie in these same sites. 352 May 49

A new primer extension analysis is used to determine the methylation pattern over the lac UV5 promoter when dimethyl sulfate is added to growing Escherichia coli. The high-resolution analysis reveals altered methylation of 15 bases when the transcription machinery occupies the promoter inside the cell and shows a striking dichotomy in the distribution of methylated bases. Four protected guanosines lie on the side of the helix shown previously to be closely bound by RNA polymerase in vitro [Siebenlist, U., Simpson, R. B., & Gilbert, W. (1980) Cell (Cambridge, Mass.) 20, 269-281]. By contrast, the 11 hyperreactive bases lie on the side of the DNA directly opposite from that bound by protein. Those not in the melted region form two distinct "back-side" patches near -35 and -16. We suggest that such hyperreactive patches can be caused by proteins bending the DNA toward themselves to allow a full range of contacts, thus distorting the helix grooves on the "back" side and facilitating attack by the methylating reagent. This leads to a proposal for the formation of transcription complexes in which RNA polymerase interacts with deformed and torsionally stressed DNA.
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PMID:High-resolution analysis of lac transcription complexes inside cells. 353 43

The interaction between E. coli RNA polymerase and the tetR promoter from pSC101, was studied by protection and premodification experiments, using dimethyl sulfate, methylation of single stranded cytosines, and DNAase I footprinting. Whereas qualitative and quantitative results from the chemical approach conform to patterns already displayed by other promoter systems, hypersensitive sites to DNAase I attack differ from those of other promoters. Distribution and nature of the contacts suggest that regions of the promoter sequence participates differently in complex formation. The involvement of major and minor grooves of the double helix in the complex with the enzyme, differs along the promoter. After a comparison of the results from seven different promoters, a pattern of conserved contacts seem to appear. Comparison of temperature dependence of local unwinding around the transcription start site (detected by the appearance of single stranded cytosines), and DNAase I footprinting, reveals that the process leading to stable complex formation can be achieved without disruption of base-pairing.
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PMID:Interaction between E. coli RNA polymerase and the tetR promoter from pSC101: homologies and differences with other E. coli promoter systems from close contact point studies. 396 Jul 16

Evidence of the presence of messenger ribonucleic acid (mRNA) in dormant spores of Bacillus subtilis has been obtained. The bulk RNA from spores was isolated and labeled in vitro with tritiated dimethyl sulfate. The spore RNA hybridized to 2.4 to 3.2% of the B. subtilis genome. The RNA hybridized to both the complementary heavy and light fractions of deoxyribonucleic acid (DNA). Bulk RNA from log-phase cells competed with virtually all the spore RNA for the heavy DNA fraction and with part of the spore RNA for the light DNA fraction. Bulk RNA from stage IV cells in sporulation also competed with all of the spore RNA for the heavy DNA fraction and with essentially all the spore RNA for the light DNA fraction. These results indicate that dormant spores contain mRNA species present in both log-phase cells and stage IV cells of sporulation. The RNA polymerase in the developing forespore must be able to recognize promotor sites for both log-phase and sporulation genes.
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PMID:Messenger ribonucleic acid of dormant spores of Bacillus subtilis. 421 60

We have examined the effect of the delta subunit on the interaction of the Bacillus subtilis RNA polymerase with an early gene promotor of phase SP82. Methylation by dimethyl sulfate, used to probe close approaches of polymerase to purines, revealed that noninitiated complexes formed by holo-enzyme (core-sigma-delta) had significantly fewer contacts than complexes formed by core-sigma. The presence or absence of delta had little or no effect on close approaches to purines in initiated complexes. DNAase I footprinting indicated that core-sigma was bound to the same region regardless of whether delta, initiating nucleotides, or both, were present. These data support the conclusion that delta acts prior to initiation to enhance promoter selectivity by limiting the number of possible interactions that the polymerase can make with DNA.
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PMID:The effect of the delta subunit on the interaction of Bacillus subtilis RNA polymerase with bases in a SP82 early gene promoter. 628 15

A method based on the differential rate of cytosine methylation in single- and double-stranded nucleic acids by dimethyl sulfate [Peattie, D.A. & Gilbert, W. (1980) Proc. Natl. Acad. Sci. USA 77, 4679-4682] has been developed for probing unpaired cytosines in DNA and DNA-protein complexes at the sequence level. Application of the method to the complexes between Escherichia coli RNA polymerase (EC 2.7.7.6) and three related promoters, lac UV5, trp, and a hybrid promoter tac resulting from the fusion of the two, reveals distinct differences in the way RNA polymerase unpairs DNA in these promoters. No single-stranded region is detectable in the complex with the trp promoter. For the lac UV5 promoter, the cytosines at positions -6, -4, -2, and -1 are in an unpaired region. The same cytosines in the tac promoter, which is homologous in sequence to lac UV5 in this region, are also found to be single stranded. For the pair of promoters lac UV5 and tac, the cytosine methylation reaction has also been used to demonstrate the steep temperature dependence of opening of base pairs by RNA polymerase. One striking feature is that the midpoint of this transition for the tac promoter is 3 degrees C lower than the corresponding value for lac UV5, even though the sequence of the unpaired region in the two promoters is identical.
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PMID:Mapping of single-stranded regions in duplex DNA at the sequence level: single-strand-specific cytosine methylation in RNA polymerase-promoter complexes. 657 69

Hedamycin forms a stable complex with DNA and introduces alkali labile linkages in the DNA. These labile linkages are located at deoxyguanosine residues and are cleaved by the treatment used for breakage at bases alkylated by dimethyl sulfate. The reaction of hedamycin with all G residues in the chain is not uniform, and certain positions, particularily those in TG tracts, are especially reactive. The reaction of hedamycin with DNA can be inhibited by ethidium bromide, suggesting that intercalation is important in positioning the reactive group of hedamycin near to the base which is modified. The low amount of hedamycin needed to produce observable breakage, its specificity for reaction with DNA and its ability to react with DNA under mild conditions make it suitable for use as a probe of protein-DNA complexes. This was shown by the ability of lac repressor and RNA polymerase to block reaction of hedamycin with the DNA of the lac regulatory region.
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PMID:Formation of alkali labile linkages in DNA by hedamycin and use of hedamycin as a probe of protein-DNA complexes. 713 91


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