9305924 Howe Bacteriophage Mu is an excellent model system for the study of mechanisms of transcriptional regulation of gene expression. During lytic development Mu exhibits a highly regulated transcriptional cascade. Early transcription is carried out by host functions; middle transcription requires the middle operon product C. This project will involve detailed molecular analysis of the protein factors and DNA sequences involved in regulation of middle operon expression and the role these factors and DNA replication play in middle promoter (Pm) activation. I. The role of potential regulatory factors discerned from examination of the promoter DNA sequence--namely, Mu repressor and A proteins, and integration host factor--will be examined. The proteins will be assayed for their effect on in vitro transcription from Pm, for binding to Pm DNA, and for the effect of mutations which knock out the proposed binding sites. Mutations which reduce Pm expression will be isolated and examined for their effects on Mor binding. The role of the dyad symmetry element t9.2 will be assessed by assaying the effect of mutations in t9.2 on Pm transcription and by determining the effect of increased and decreased transcription at t9.2 on Pm expression. II. The DNA binding domain of the Mor protein will be localized to the amino or carboxyl terminus of Mor by making hybrid proteins between Mor and the closely related C protein and determining which end confers promoter specificity. Then, detailed cassette mutagenesis of the DNA binding domain will be carried out to isolate suppressor mutations which allow Pm promoter-down mutants to function. This analysis will indicate the amino acids involved in DNA binding and help to define whether or not Mor uses a helix- turn helix motif for binding. III. The mechanism of the replication dependence of middle transcription will be examined by analysis of the effect of DNA supercoiling on Pm transcription, identification of middle pro moter bases contacted by protein factors in dimethylsulfate (DMS) methylation protection assays, and identification of bases which become single stranded during open complex formation by potassium permanganate (KMn04) treatment. Lastly, a comparison will be made of the DMS methylation protection and KMn04 patterns of replicating and non-replicating Mu DNA to see if there is altered protein binding or DNA conformation between the two DNA forms. *** Organisms have evolved a number of regulatory mechanisms which allow them to maintain an appropriate balance of various life processes. One energy efficient mechanism is to regulate the expression of specific genes such that their products are made only at the times they are needed. Viruses provide excellent model systems for the study of these mechanisms, since their development involves the sequential expression of specific sets of genes. This project involves the molecular analysis of the mechanisms regulating a set of genes expressed at intermediate times during development of the Mu virus, which infects and grows in laboratory strains of the bacterium E. coli. The project will involve precise definition of the viral and host proteins that effect this regulation by interaction with the regulatory target sequences of the genes and identification of those target sequences and the events occurring at them which turn the genes on and off. The approach will include isolation and analysis of the effects of mutations which alter the proteins and regulatory sites as well as biochemical characterization of the events occurring under normal conditions and determination of the role that DNA conformation plays in these processes. %%%
