Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR.
We have made site-specific mutations in the promoter-operator region of the mercury-resistance (mer) operon of transposon Tn501. Mutations were selected to alter the spacing between the -10 and -35 promoter elements without altering the sequence of a 7 bp dyad symmetrical sequence, which is the site of binding of the regulatory protein, MerR. (MerR acts as a repressor in the absence of mercuric salts and as an inducer in their presence, and binds to the same site in each case). Transcription from the mutant promoters was measured in vivo in the presence and absence of MerR and of mercuric salts; and the relative affinities of the mutant promoters for partially purified MerR were determined in vitro by gel-shift assay in the presence and absence of mercuric salts. The 19 bp spacer was found to be essential for correct induction and repression of the operon; a spacer size of 20 or 21 bp prevents induction, and a spacer size of 18 or 17 bp causes the promoter to be highly active under all conditions. Double mutations, which alter the position of the 7 bp dyad relative to the -10 and -35 sequences without altering their spacing prevent induction by the MerR-Hg(II) complex, demonstrating the tight constraints on the positions of MerR and RNA polymerase in the transcriptional complex. The data are compatible with a model for induction of the mer promoter involving a local conformational change in the DNA structure caused by the MerR-Hg(II) complex.
Parkhill
J
J
Microbial Molecular Genetics and Cell Biology Research Group, School of Biological Sciences, University of Birmingham, Edgbaston, UK.
Brown
N L
NL
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004275:DNA, Ribosomal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008628:Mercury / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; C051124:MerR protein, Bacteria; D012097:Repressor Proteins; D014157:Transcription Factors; D008628:Mercury