Promoter recognition by Escherichia coli RNA polymerase. Influence of DNA structure in the spacer separating the -10 and -35 regions.
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.
D001345:Autoradiography; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D003720:Densitometry; D004926:Escherichia coli / Q000235:genetics; D008745:Methylation; D011401:Promoter Regions, Genetic; D013463:Sulfuric Acid Esters
D004269:DNA, Bacterial; D013463:Sulfuric Acid Esters; D012321:DNA-Directed RNA Polymerases; C007482:dimethyl sulfate