Deletion analysis of RNA polymerase interaction sites in the Escherichia coli lactose operon regulatory region.
Two sets of deletions produced in vitro by S1 nuclease were used to study the structure and function of promoters lacPUV5 and lacP115. The upstream boundary of the RNA polymerase binding site in lacPUV5 was determined by comparing the levels of beta-galactoside expression in vivo programmed from a set of deletions progressively extending into the -35 region of the lacPUV5 promoter. Sequences upstream from base-pair -37 are not necessary for the full functioning of lacPUV5. A deletion that removes base-pair -37 retains only half of the promoter activity. Deletion of the first T X A base-pair of the consensus -35 region sequence, 5' T-T-T-A-C-A 3', leads to a sixfold reduction of promoter activity. Deletion of the whole -35 region of lacPUV5 leads to at least a 20-fold reduction of its promoter activity. Abortive initiation assays were performed on the fully functional lacPUV5 and two lacPUV5 deletions, which removed part of the -35 consensus sequence, to study their effect on the kinetics of RNA polymerase-promoter open complex formation. These two deletions show a 3.5 to 7-fold reduction in KB. Analysis in vivo of lacP115 showed that sequence information upstream from the -35 region is important for the full functioning of lacP115. A deletion removing sequences upstream from -41 caused a three- to fourfold reduction in promoter activity, apparently due to reduced transcription initiation. lacP115 has a much lower k2 value than lacPUV5; its KB value is about threefold higher than that of lacPUV5.
D001483:Base Sequence; D001665:Binding Sites; D002872:Chromosome Deletion; D004269:DNA, Bacterial; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D007700:Kinetics; D007763:Lac Operon; D009154:Mutation; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger; D012321:DNA-Directed RNA Polymerases