Interdisciplinary studies of the mechanisms for transcriptional activation and metal-specific gene expression in the bacterial mercuric ion resistance operon(mer) are described. Both objectives center on characterization of the interaction of the MerR metalloregulatory protein with toxic heavy metals and with biopolymers in the genetic control apparatus. MerR represses the expression of the mer genes in the absence of Hg(II) and activates transcription by E.coli RNA polymerase when Hg(II) concentrations exceed 10 nM. Both activities are manifest while MerR is bound at a single site on the DNA. Preliminary results indicate an unprecedented role for DNA distortion in the mechanism for positive control of transcription. A combination of transcriptional initiation kinetic studies, novel footprinting strategies, biophysical measurement of equilibrium sedimentation and binding experiments, NMR and microscopy will be used to deconvolute the topology of the transcription complex, the energetics of the switching process and the molecular mechanism of transcriptional activation. Positive control mechanisms are poorly understood and yet are of fundamental importance in understanding the molecular basis of genetic regulation and its role in human-health. The molecular basis of heavy metal recognition in this system will be probed at the biopolymer and coordination chemistry levels. Physical-inorganic studies of Hg(II) interaction with MerR will involve, electronic spectroscopy, 199Hg Solid state and solution NMR experiments, synthesis and characterization of mercuric ion complexes with imidazolate and alkyl thiolate ligands. The MerR system can serve as a paradigm for metal-responsive regulation in prokaryotic and eukaryotic heavy-metal homeostasis systems.
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