In this application, studies are proposed to investigate the regulation of replication gene expression. We will focus on the dnaE gene (structural gene for the alpha subunit of the DNA polymerase Ill holoenzyme) and the dnaX gene (encodes the gamma and tau subunit genes). In the last grant period it was demonstrated that the dnaE gene was in a complex operon that covered over 14,000 bases and contained the structural genes for eleven proteins, many involved in DNA metabolism and membrane biogenesis. The operon, named the Macromolecular Synthesis II operon (MMS II) displays the capacity for complex discoordinate regulation. It contains at least eight promoters and an internal intercistronic sequence that is the target for an endoribonuclease. This cleavage may be of regulatory importance. Evidence has also been gathered for translational coupling. We will quantitate the importance of each internal promoter in terms of the total alpha subunit synthetic contribution so that we will be able to set priorities for the most important promoters to pursue. The effectors of dnaE and X expression will be determined by a combined in vivo and in vitro approach. We will also determine which factors regulate frameshifting in dnaX, a mechanism that determines the balance of gamma vs. tau subunits in the cell. We will also perform experiments to explore the nature of some intriguing regulatory circuits between holoenzyme subunits and membrane biogenesis genes, particularly lipoproteins.
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