The proposed research program is in the general area of mechanisms for regulating the activity of genes. Its broad and practical implications are for understanding gene regulation in developing systems and infectious processes, and for precise interventions concerning gene activity. The particular system involves a bacterial virus, whose development is determined by a programmed sequence of gene expression. The virus codes for a chromatin-forming protein, called TF1, which is a homologue of the ubiquitous bacterial chromatin-forming proteins, called HU or type 11 DNA-binding proteins. The proposed research program pursues two lines of work. The first group of studies deals with the genetics, function, structure and DNA binding properties of TF1, by: 1) generating temperature-sensitive TF1 by mutagenesis targeting the entire TF1 gene, selectively; 2) analyzing defects of viral development, caused by the failure of TF1 function, at the molecular level; 3) analyzing structure- function relationships in TF1 by generating mutant TF1 with specific, appropriately chosen changes of amino acid sequence and analyzing the consequences of these changes for the affinity and specificity of DNA binding; 4) engaging in collaborations, involving X-ray crystallography and NMR spectroscopy, to determine the 3-dimensional structure of TF1 and of TF1-DNA complexes. A second group of in vivo and in vitro studies deals with the mechanism of selectively shutting off transcription at some, but not all, SP01 middle promoters by: 1) mapping and sequencing middle promoters of each regulatory subtype, seeking to detect distinctive conserved sequences that may correlate with one regulatory class or another: 2) utilizing the ability to clone in phage SP01 for analyzing promoter strength and regulation in vivo by molecular genetic methods; 3) analyzing overlapping late/middle specificity transcription initiation with appropriate in vitro systems and precise 5'-end mapping; 4) analyzing the occupancy of promoters by proteins in vivo; 5) examining effects of topoisomerase inhibitors on regulation at specific middle and late promoters; 6) examining the effect of DNA supercoiling on in vitro transcription at selected middle and late promoters; 7) analyzing the properties of virus-coded RNA polymerase-binding proteins in suitable in vitro systems. Two subsidiary projects are also proposed: one concerns hybrid TF1 molecules, and the other concerns gene expression at a viral gene regulation-specific promoter on a resident plasmid.