The goal of the proposed research is to determine the mechanisms underlying the control of transcription of the Escherichia coli glyA and gcv operons, which encode enzymes necessary for the formation of the one-carbon units essential for cellular methylation reactions. These systems are under both positive and negative control by multiple regulatory proteins. This complexity reflects the cell's flexibility in balancing production of the one-carbon substrates by the end products of the pathways using these compounds. The experiments proposed will examine the interplay of the four known activator proteins and two known repressor proteins with their respective DNA target sites, with RNA polymerase, and with each other for controlling transcription initiation. A combination of genetic and biochemical techniques will be used. The structural genes (glyA and gcvTHP) and the known regulatory genes (gcvA, gcvR, purR, metR, crp, and lrp) have been cloned and sequenced. Translational fusions to the lac operon have been constructed to facilitate in vivo studies. In vitro-produced and genetically-selected mutations will be isolated to define the functional domains of the regulatory proteins involved. In vitro DNA binding assays and footprinting assays will be used to measure the relevant binding constants for individual regulatory proteins and for combinations of regulatory proteins to examine possible positive and negative cooperativity in binding. The regulatory systems will be reconstituted first in part and then in entirety in in vitro transcription and abortive initiation assays to examine how the regulatory proteins influence transcription initiation by RNA polymerase. An understanding of glyA and gcv regulation, where pathway specific and global-acting regulatory proteins interact to regulate transcription, will provide information concerning the fundamental rules that govern the protein-protein and DNA-protein interactions controlling transcription initiation.
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