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Abstract
Various aspects of the induction of the mer promoter of TN501 by the MerR repressor/activator were investigated. Specific insertion and deletion mutations in the mer promoter were produced, and the effect of MerR on their ability to direct transcription was analysed. Insertion of a single base pair between the -10 and -35 elements of the promoter severely reduces induction by MerR-Hg(II) and prevents repression, although MerR binding is not affected. Insertion of two base pairs prevents both induction and repression by MerR. Deletion of one or two base-pairs within the promoter causes it to direct transcription at a level similar to that of the fully-induced wild-type promoter, but in the absence of MerR. The promoter containing deletions are repressed by MerR in both the presence and absence of mercury. A constitutively inducing MerR mutant (MerR*As) was constructed by site-specific mutagenesis and was shown to cause a distortion of the promoter DNA in both the presence and absence of mercury, similar to that produced by the w.t. protein in the presence of mercury. The degree of distortion of the DNA was shown to be correlated with the degree of induction of the mer promoter. The kinetics of activation of the mer promoter by MerR and HgCl2 were analysed in vivo and compared to the published results for a reconstituted in vitro system. It was established that the mer promoter responds in vivo to changes in mercury concentration in an ultrasensitive manner. MerR binding to sequences in Tn501 other than the mer promoter was investigated, but no additional binding sites were found. Mutants of MerR were isolated which confirmed the identity of the putative DNA binding helix-turn-helix region. The results of these investigations are discussed in terms of a model for the mechanism of transcriptional activation by MerR that involves a protein-directed distortion in DNA structure. This mechanism is unique amongst known transcriptional activators.
