The overall objective of this work is to understand the genetic mechanisms that regulate synthesis of the replication DNA polymerase of bacteriophage T4 and its interactions with other proteins of the T4 DNA replication complex. Of particular interest to us is the mechanism by which the polymerase regulates its own synthesis in vivo. We want to find out if this regulation occurs at the transcriptional or translational levels. Also, we plan to fuse different NH2-terminal segments of the structural gene for T4 DNA polymerase (T4 gene 43) to an E. coli lacZ (beta- galactosidase) gene in order to effect the synthesis of chimeric proteins that harbor activities associated with different N2H- terminal lengths of the single polypeptide (896 amino acid) T4 gene 43 product. A coupled transcription/translation cell-free assay system and purified transcription and translation assays will be used to reconstruct the mechanism of autogenous repression by T4 DNA polymerase in vitro and to localize the target site (operator) for repression. The roles of DNA polymerase accessory proteins in control of gene 43 expression will also be tested in these in vitor assays. The proposed studies may reveal that T4 DNA polymerase biosynthesis is coordinately controlled by availability of other replication proteins (global regulation of the replication complex). Once the target (operator) for gene 43 autogenous repressor is identified, it will be altered by in vitro manipulations in order to identify nucleotide residues important in recognition of the operator by T4 DNA polymerase and added corepressors. The recombinant proteins synthesized by gene 43- lacZ fusions will be characterized for in vivo properties and purified and assayed for nucleic-acid-binding and repressor activities in vitro. Antibodies will be generated to different protein segments of the T4 DNA polymerase and used for purification of and analysis of activities intrinsic to mutant peptides.
