9604653 Howe This investigator's model system for regulation of gene expression is the middle promoter Pm of the Mu virus which infects Escherichia coil K12. The middle transcript is made by the host RNA polymerase, but only in the presence of the Mu activator protein Mor and DNA replication. Studies during the previous grant period revealed that Mor activates Pm by binding as a dimer upstream of and overlapping the -35 region, interacting with both the alpha and sigma subunits of RNA polymerase, and thereby generating a DNA distortion which facilities open complex formation and transcription initiation. In the absence of DNA replication, Mor does not bind and open complexes do not form. The current project includes experiments (i) to define at a molecular level the specific roles of the sigma and alpha subunits of RNA polymerase, by using footprinting and in vitro transcription assays with wild-type and mutant forms of Pm, alpha, and sigma; (ii) to identify the amino acids in Mor which are required for Pm binding and interaction with alpha and sigma, by assaying the effect of targeted alanine substitutions in Mor on these processes; (iii) to determine the origin and role of the DNA distortion by mutational analysis of nearby sequences and identification and characterization of host proteins which may participate in distortion formation; and (iv) to determine whether there is a host "repressor" protein which binds to non-replicating Pm DNA, thereby preventing Mor binding, by identification of Pm-binding proteins and characterization of Pm and host mutations conferring replication independence. This research will define the molecular mechanism by which the Mu middle genes are regulated---that is, how they are kept silent prior to the time in development when they are needed and how they are turned on at the appropriate time. Not only will these results provide an understanding of these novel mechanisms for turning genes on and off, but those mechanisms can serve as models for studying and understanding the regulatory mechanisms occurring at other important viral, bacterial, animal cell and human genes. Since much of any organism's ability to maintain an appropriate internal milieu, respond to environment stimuli, and carry out the programmed changes we call development depend on its ability to regulate expression of specific genes, this research is of great importance. In addition the experiments will serve as an excellent, state-of-the-art, training experience in genetics, biochemistry, and molecular biology for the graduate students who participate in them.
