GENETIC-ANALYSIS OF THE TN21 MER OPERATOR-PROMOTER

The mercury resistance operon, mer, of the transposon Tn21 is transcri bed from two overlapping divergent promoters: P(R) for the regulatory gene, merR, and P(TPCAD) for the structural genes, merTPCAD. Transcrip tion of merTPCAD is repressed in the absence of Hg(II) and activated i n 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 bet ween the -35 and -10 hexamers of P(TPCAD). Analysis of the expression of transcriptional fusions to hydroxylamine- and oligonucleotide-gener ated mutants of this divergent operator-promoter region identified key bases involved in MerR-dependent repression of P(TPCAD) and of P(R) a nd in activation of P(TPCAD). Six of the seven mutants affecting the p alindromic region were altered in their ability to bind the MerR prote in in vitro as measured by fragment retardation assays. These differen ces in in vitro MerR binding correlated well with the in vivo measurem ents of repression or of activation. Bases identified as functionally relevant by this genetic analysis coincide extensively with those prev iously identified as relevant via in vivo footprinting. Four major poi nts emerge from this analysis: (i) transition and transversion mutatio ns within the spacer between the -10 and -35 hexamers of P(TPCAD) gene rally have little effect on the MerR-independent (i.e., unregulated) e xpression of either promoter; (ii) alteration of certain bases in the MerR-binding dyad affects repression of P(TPCAD) differently than repr ession of P(R); (iii) certain dyad changes can impair activation of P( TPCAD) more severely than repression of this promoter; and (iv) mutati ons in the -10 hexamer of P(TPCAD) which also effect P(R) expression d efine one of two potential -10 hexamers in P(R) as actually functional in vivo.