Through the use of natural template probes, many of the components of the E. coli replicative machinery have been isolated. These components include a priming enzyme, a single- stranded DNA binding protein and the complex DNA polymerase III holoenzyme that consists of at least seven different subunits. Much progress has been made in assigning functional roles to these proteins and in describing their mechanism of action. However, less is known of their regulation or the coordination of their synthesis from disparate regions of the E. coli chromosome. This project proposes to investigate how the synthesis of replication proteins is regulated and, ultimately, how this regulation is coordinated with the overall control of cell growth and division. During the proposed grant period, we plan to focus primarily on the regulation of the expression of two key replication genes, dnaE, the structural gene for the central catalytic subunit of the replicative complex, and dnaZX, an essential gene that encodes two critical auxiliary proteins. We have just completed sequencing these two genes. These studies will be extended by (i) mapping the regions transcribed with these two genes, (ii) determining how one open reading frame in dnaZX encodes two holoenzyme subunits, (iii) describe more fully the regulatory response of holoenzyme structural gene expression at both the mRNA and protein level to changes in growth and environmental conditions, (iv) extend these regulatory studies using operon and protein fusions with lacZ, (v) prepare additional monoclonal antibodies against holoenzyme subunits to support our regulatory studies and (vi) develop and in vitro protein synthesizing system to assay for specific effectors of replication protein transcription and translation.
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