Site specific DNA binding proteins play important roles in the regulation of transcription; the replication and repair of DNA; phage and viral DNA packaging; site-specific recombination and transposition, and restriction-modification. One of the goals of modem molecular biology is to understand these processes in molecular detail. A true understanding of DNA recognition would allow the design of proteins that could bind to specific sequences in cellular or viral DNA. If such proteins could be targeted to appropriate cells, they might be used to modulate gene expression or viral growth and thus be valuable in the treatment of disease. The objective of the research described in this proposal is to understand the relationship between the sequences and structures of repressor proteins and their DNA recognition and gene regulatory activities . As experimental systems, three phage repressors, P22 Arc, P22 Mnt, and the N-terminal domain of phage lambda repressor will be studied. Arc and Mnt are small, homologous proteins that use the recently discovered beta-ribbon motif for DNA recognition. Lambda repressor is a helix-turn-helix DNA binding protein. Each of these systems is readily accessible to study by a combination of protein and nucleic acid chemistry, molecular genetics analysis, kinetic and equilibrium studies, and X-ray crystallography, thereby allowing meaningful structure-function studies. X-ray crystallographic studies of protein-DNA complexes will be combined with biochemical studies of the effects of side chain substitution mutations on the free energy and specificity of DNA recognition to determine the mechanisms by which remarkable specificity changes in DNA binding can be caused by single amino acid changes. The mechanism by which Arc prevents or slows the isomerization of RNA polymerase during transcription will also be addressed by a combination of biochemical and genetic studies. Optimal DNA binding sites will be determined for Arc in three different structural backgrounds to determine if quaternary structure constrains idealized DNA contacts and vice versa.