title: In vivo DNA-protein interactions at the divergent mercury resistance (mer) promoters. II. Repressor/activator (MerR)-RNA polymerase interaction with merOP mutants.
abstract: Transcription of the Tn21 mercury resistance (mer) operon is regulated by MerR which represses and activates the mer structural genes (merTPCAD) in the absence and presence of Hg(II), respectively. The promoter for the structural genes (PTPCAD) is divergently overlapped with the promoter for the regulatory gene (PR), and a dyadic operator lies between the -10 and -35 hexamers of PTPCAD. Using in vivo dimethyl sulfate and KMnO4 footprinting of mutant mer operator-promoter (merOP) DNA to observe MerR and RNA polymerase-mediated interactions with the merOP region, we have identified three distinct domains within the palindromic mer operator. Dyad domain I consists of the outermost bases on the left arm of the operator palindrome whose alteration causes a shift, but apparently not a major loss, in occupancy by MerR, and no decrease in RNA polymerase occupancy. Mutants in dyad domain I are semiconstitutive but support additional Hg(II)-induced open complex formation at PTPCAD. Dyad domain II consists of the four highly conserved inner bases ( ... GTAC ... GTAC ... ) of the seven-base interrupted dyad, alteration of which severely modifies both MerR and RNA polymerase contacts in the promoter region. Mutants in domain II generally allow constitutive open complex formation at PR. One unusual mutant of this group retains most of the wild-type dyad's ability to repress both promoters but is unable to support activation at PTPCAD in response to Hg(II), indicating that MerR undergoes a conformational change and that the required base contacts for activation are different than those for repression. Dyad domain III is tentatively defined by a mutant in the outermost base of the right palindrome arm which is unaffected in either MerR or RNA polymerase occupancy, however, a second lesion within the PTPCAD -10 hexamer of this mutant limits effective open complex formation. Other mutations lying solely within the -10 RNA polymerase recognition hexamer of PTPCAD are similarly competent in both MerR and RNA polymerase binding, but inadequate for open complex formation. One such mutant also affects the overlapping -10 hexamer of PR and results in reduced occupancy by both MerR and RNA polymerase, likely as a result of inefficient transcriptional initiation of merR mRNA. Finally, mutations affecting the -35 hexamer of PTPCAD bind MerR but not RNA polymerase.(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Microbiology, University of Georgia, Athens 30602-2605.
authors.forename: I W
authors.initials: IW
authors.lastname: Lee
authors.forename: V
authors.initials: V
authors.lastname: Livrelli
authors.forename: S J
authors.initials: SJ
authors.lastname: Park
authors.forename: P A
authors.initials: PA
authors.lastname: Totis
authors.forename: A O
authors.initials: AO
authors.lastname: Summers
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000378:metabolism; 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; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D009875:Operator Regions, Genetic; D009876:Operon; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: 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