Genetic analysis of the Tn21 mer operator-promoter

Abstract

The mercury resistance operon, mer, of the transposon Tn21 is transcribed from two overlapping divergent promoters: PR for the regulatory gene, merR, and PTPCAD for the structural genes, merTPCAD. Transcription of merTPCAD is repressed in the absence of Hg(II) and activated in the presence of Hg(II) by the regulatory protein, MerR. In addition, MerR represses its own expression regardless of the presence of Hg(II). MerR binds as a dimer to a single region of dyad symmetry lying between the -35 and -10 hexamers of PTPCAD. Analysis of the expression of transcriptional fusions to hydroxylamine- and oligonucleotide-generated mutants of this divergent operator-promoter region identified key bases involved in MerR-dependent repression of PTPCAD and of PR and in activation of PTPCAD. Six of the seven mutants affecting the palindromic region were altered in their ability to bind the MerR protein in vitro as measured by fragment retardation assays. These differences in in vitro MerR binding correlated well with the in vivo measurements of repression or of activation. Bases identified as functionally relevant by this genetic analysis coincide extensively with those previously identified as relevant via in vivo footprinting. Four major points emerge from this analysis: (i) transition and transversion mutations within the spacer between the -10 and -35 hexamers of PTPCAD generally have little effect on the MerR-independent (i.e., unregulated) expression of either promoter; (ii) alteration of certain bases in the MerR-binding dyad affects repression of PTPCAD differently than repression of PR; (iii) certain dyad changes can impair activation of PTPCAD more severely than repression of this promoter; and (iv) mutations in the -10 hexamer of PTPCAD which also effect PR expression define one of two potential -10 hexamers in PR as actually functional in vivo.