The primary goal of this proposal is to elucidate the mechanism of action of a model antiterminator, the N protein encoded by temperate phage lambdal. N is a small, modular protein that interacts with a specific genetic signal (called nut) on the phage genome and a number of host proteins (called Nus factors) to modify host RNA polymerase to a termination-resistant mode. The current working model proposes that an arginine motif present in N binds the boxB RNA hairpin encoded by nut, bringing N in close contact with the target polymerase. N then captures polymerase through mRNA looping and collaboration of an adapter host factor (NusA), and leads RNA polymerase through downstream terminators by being an operon-specific subunit. Additional host factors interact with conserved RNA signals and N to facilitate both the assembly as well as persistent antitermination. The proposed experiments will combine genetic, biochemical and biophysical approaches to accomplish several major objectives. The first goal is to carry out several lines of mechanistic studies to rigorously test various predictions of the model, further dissecting the role of the core components in termination-suppression and elucidating the underlying molecular mechanisms. The second goal is to isolate the missing host factor(s) and their genes by independent genetic and biochemical approaches and elucidate their function in assembly of the N-antitermination complex, persistent antitermination, and in switching phage development from lysogenic to the lytic mode. The third goal is to study N-boxB interaction with molecular genetic and biophysical approaches to decipher the chemical principles that govern RNA-protein interaction. The fourth goal, a new direction of the project, is to identify cellular genes that are activated by N-like antitermination mechanism, and isolate potential cellular homologues of N. These studies should provide fundamental information on the structure, function and regulation of the prokaryotic transcription apparatus, should illuminate the function of RNA signals in transcription control, and should reveal aspects of the molecular basis of sequence-specific recognition of RNA signals by regulatory proteins.
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