MUTATIONS IN THE ALPHA AND SIGMA-70 SUBUNITS OF RNA-POLYMERASE AFFECTEXPRESSION OF THE MER OPERON

The mercury resistance (mer) operon is transcribed from overlapping, d ivergent promoters: P-R for the regulatory gene merR and P-TPCAD for t he structural genes merTPCAD. The dyadic binding site for MerR lies wi thin the 19-bp spacer of the sigma(70)-dependent P-TPCAD. Unlike typic al repressors, MerR does not exclude RNA polymerase from P-TPCAD but r ather forms an inactive complex with RNA polymerase at P-TPCAD prior t o addition of the inducer, the mercuric ion Hg(II). In this ''active r epression'' complex, MerR prevents transcriptional initiation at merTP CAD until Hg(II) is added, When Hg(II) is added, MerR remains bound to the same position and activates transcription of merTPCAD by distorti ng the DNA of the spacer region, MerR also represses its own transcrip tion from P-R regardless of the presence or absence of Hg(II), To expl ore the role of MerR-RNA polymerase in these processes, we examined mu tations in the sigma(70) and alpha subunits of RNA 70 polymerase, muta tions known to influence other activators but not to impair transcript ion generally, We assessed the effects of these sigma(70) and alpha mu tants on unregulated P-TPCAD and P-R transcription (i.e., MerR-indepen dent transcription) and on the two MerR-dependent processes: repressio n of P-TPCAD and of P-R and Hg(II)-induced activation of P-TPCAD Among the MerR-independent effects, we found that mutations in regions 2.1 and 4.2 of rpoD suppress the deleterious effects of nonoptimal promote r spacing, Some C-terminal rpoA mutants also have this property to a c onsiderably lesser degree, Certain ''spacer suppressor'' variants of r poA and of rpoD also interfere with the MerR-dependent repression of P -TPCAD and P-R. MerR-Hg(II) -mediated transcriptional activation of P- TPCAD was also affected in an allele-specific manner by substitutions at position 596 of sigma(70) and at positions 311 and 323 of alpha. Th us, certain changes in sigma(70) or alpha render them either more or l ess effective in participating in the topologically novel transcriptio nal control effected by MerR at the divergent mer operons.