In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. I. Metalloregulatory protein MerR mutants.
Regulation of transcriptional initiation of the Tn21 mercury resistance (mer) operon occurs at the divergent promoter region lying between the structural genes (merTPCAD) and a regulatory gene (merR). During repression, both promoters are negatively regulated by MerR bound to a dyadic operator located between the -10 and -35 hexamers of PTPCAD. Upon Hg(II) induction, MerR activates transcription only from PTPCAD and continues to repress transcription from PR. Using in vivo dimethyl sulfate and KMnO4 footprinting of the merOP region of strains carrying wild-type MerR or MerR mutants, we have dissected the steps in MerR-mediated positive and negative regulation of the divergent mer promoters. The greater sensitivity of primer extension footprinting allowed the resolution of details previously undetectable in vivo. Two MerR mutants unable to bind merOP DNA allow RNA polymerase to form an open complex preferentially at PR. The intensity of the PR open complex is considerably less than that which occurs upon MerR-Hg(II) activation of PTPCAD and considerably greater than that which occurs at PTPCAD when MerR is deleted; this is the first in vivo estimate of the relative strengths of these two promoters. Although retaining the wild-type capacity to sequester RNA polymerase in the closed complex, the four MerR mutants defective in Hg(II) binding do not distort the dyad DNA or foster open complex formation when the inducer is added. Two activation-defective MerR mutants foster closed complex formation as well as wild-type, but they do not distort the dyad DNA or foster open complex formation when the inducer is added, although they are also able to bind Hg(II). Two semiconstitutive inducible MerR mutants differ from each other in that one (which lies nearer the COOH terminus) does distort the dyad center upon Hg(II) induction, whereas the other (which lies in near the center of merR) exhibits no dyad distortion upon induction. Paradoxically, despite their relatively strong semiconstitutive expression of mer-lac transcriptional fusions, neither of these mutants has a detectable open complex in the absence of added Hg(II).
Livrelli
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Department of Microbiology, University of Georgia, Athens 30602.
Lee
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Summers
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004352:Drug Resistance, Microbial; D015964:Gene Expression Regulation, Bacterial; D008628:Mercury / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009875:Operator Regions, Genetic; D009876:Operon; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012097:Repressor Proteins; D012321:DNA-Directed RNA Polymerases; D008628:Mercury