We will study metal-responsive gene regulation in the bacterial plasmid- determined mercury (Hg) resistance (mer) operon. The mer locus is very widely found in eubacteria and mediates detoxification of inorganic and organic Hg compounds. The striking conservation of Hg binding motifs of mer proteins in the proteins altered in the human copper storages diseases (Menkes' and Wilson's diseases) underscores the importance of mer as a model for understanding human exposure to toxic metals. Moreover, acute or chronic exposure of an animal host to mercury compounds results in the proliferation in the normal flora of bacteria which carry the mer locus. In the bacterium, expression of the mercurial detoxification genes is controlled by a novel Hg-binding transcriptional regulator, MerR. Mercury-responsive regulation of gene expression in this system is, thus, a model for regulation of genes by transition metals and has been found to have several unusual aspects not seen in gene regulation by non-metallic metabolites. (l) We will use molecular genetics to probe the interactions of wildtype and mutant merR proteins with the mer operator-promoter region and with RNA polymerase in order to establish the molecular basis for transcriptional repression and activation. (2) We have discovered that mer expression is also controlled by premature transcriptional termination (i.e. mer has a very efficient mid-operon transcriptional attenuator). We will use genetics to dissect the elements of this attenuator and identify and characterize the cis- and trans-acting agents involved. (3) We have recently discovered a surprising role for the host cell hydroperoxidases (including catalase) in the expression of Hg resistance and will define this role and explore other host chromosomal genes important in Hg detoxification. (4) We will also begin a new project on the interaction of MerR with metal ions by isolating merR mutants which recognize metals other than Hg and by characterizing their purified protein products using NMR, ESR, and resonance Raman spectroscopy.
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