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)
Highlights
Transcription of the Tn21 mercury resistanc(emer) polymerase binding, but inadequate for open complex operon is regulated by MerR which represses and ac- formation
Mutants in Transcription of the mer operon inTn21andTn501 is dyad domain I are semiconstitutive but support addi- positively and negatively regulated by a DNA-bindingprotein, tional Hg(I1)-induced open complex formation a t MerR, which binds to a region of dyad symmetry within the
) PTPCADDy.addomain conserved inner bases consists of the four highly .GTAC.. .GTAC.. of the mer operator-promoter region (1, 2). This dyadic region is located between the -10 and -35 hexamers of the seven-base interrupted dyad, alteration of which se- structural gene promoter, PTPCAD, which isdivergently oriverely modifies both MerR and RNA polymerase con- ented relative to the merR promoter, PR(Fig. 1).The 1-bp’
Summary
In Viuo Methylation of DNA-Cell culture, DNA methylation, and plasmid extractions were as previously reported (1,19). EDTA, 0.1 M NaC1, pH 8.0) into the culture, andthe cells were washed once with 1 ml of TEN buffer Plasmid Extraction and Fragmentation-Plasmid DNA was purified as described by Heltzel et al (1).DMS- and KMn04-modified plasmid DNAs were incubated with 100 r l of 1M piperidine at 90 "C, and the fragmented DNA was subsequently used as the template for primer extension. All the experiments were repeated a t least twice for each strain, and only bands consistently more than 30%darker or lighter than the corresponding band in the in vitro methylated G lane were designated as enhanced or protected, respectively. Other bands may appear to vary in a single specific gel, we discuss below only those bands which reproducibly gave the same signal in all experiments
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