The objectives of this proposal are to explore the factors involved in the regulation of expression of bacteriophage T4 DNA replicative proteins and to probe structure-function relationship of T4 polymerase. The experiments proposed here should provide new insights into the mechanisms used to coordinate DNA replication with the balanced synthesis of DNA replication proteins, and about important functional domains of T4 DNA polymerase, through structural characterization of DNA polymerase mutants. The DNA replication region of the T4 genome contains gene 43 (DNA polymerase), genes 44, 45 and 62 (polymerase accessory proteins) and the regA gene (translational repressor). The product of the regA gene controls the expression of genes 44, 45 and 62, as well as a number of other T4 prereplicative genes, including the regA gene itself, at the level of translation. To determine the mechanism of this translational repression, we will purify regA protein from available overproducing strains, and test its ability to bind to mRNA (as judged from fluorescence quenching assays) and to inhibit in vitro translation of gene 45, gene 44 and gene 62. The target site for regA action will be examined by deletion analysis of the gene 45 mRNA leader region, and by mutagenesis of genes 45 and 44. To examine the mechanism of autogenous regulation of regA, the possibility of translational coupling of genes 44, 62 and regA will be examined. If the three genes are co-translated, then experiments will be done to determine if there is a regA target site at the beginning of each gene or at a single site at the 5' end of the polycistronic mRNA. Previous studies have suggested that there are functional domains of T4 DNA polymerase which are detectable by characterization of mutant DNA polymerases. Accordingly, we plan to determine the nucleotide changes present in four particularly interesting T4 DNA polymerase mutants.
