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. 1997 Mar;179(5):1787–1795. doi: 10.1128/jb.179.5.1787-1795.1997

Mutations in the alpha and sigma-70 subunits of RNA polymerase affect expression of the mer operon.

L F Caslake 1, S I Ashraf 1, A O Summers 1
PMCID: PMC178895  PMID: 9045842

Abstract

The mercury resistance (mer) operon is transcribed from overlapping, divergent promoters: PR for the regulatory gene merR and P(TPCAD) for the structural genes merTPCAD. The dyadic binding site for MerR lies within the 19-bp spacer of the sigma70-dependent P(TPCAD). Unlike typical repressors, MerR does not exclude RNA polymerase from P(TPCAD) but rather forms an inactive complex with RNA polymerase at P(TPCAD) prior to addition of the inducer, the mercuric ion Hg(II). In this "active repression" complex, MerR prevents transcriptional initiation at merTPCAD until Hg(II) is added. When Hg(II) is added, MerR remains bound to the same position and activates transcription of merTPCAD by distorting the DNA of the spacer region. MerR also represses its own transcription from PR regardless of the presence or absence of Hg(II). To explore the role of MerR-RNA polymerase in these processes, we examined mutations in the sigma70 and alpha subunits of RNA polymerase, mutations known to influence other activators but not to impair transcription generally. We assessed the effects of these sigma70 and alpha mutants on unregulated P(TPCAD) and PR transcription (i.e., MerR-independent transcription) and on the two MerR-dependent processes: repression of P(TPCAD) and of PR and Hg(ll)-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 promoter spacing. Some C-terminal rpoA mutants also have this property to a considerably lesser degree. Certain "spacer suppressor" variants of rpoA and of rpoD also interfere with the MerR-dependent repression of P(TPCAD) and PR. MerR-Hg(II)-mediated transcriptional activation of P(TPCAD) was also affected in an allele-specific manner by substitutions at position 596 of sigma70 and at positions 311 and 323 of alpha. Thus, certain changes in sigma70 or alpha render them either more or less effective in participating in the topologically novel transcriptional control effected by MerR at the divergent mer operons.

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Selected References

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