This grant has supported study of both transcription and chaperone function, and now is concentrated on transcription - at this stage, mainly the mechanism of transcription termination mediated by a specific bacteriophage protein, Nun, and also the function of an essential cellular protein NusG that is (at least) an accessory to rho- dependent termination. 1) NusG affects both termination efficiency at rho-dependent terminators and the related processes of lambda N-mediated antitermination and Nun-mediated termination (see below). Nun is thought to interact with other N accessory factors, like NusA. Dr. Gottesman plans to use a Nun-resistant NusG (NusG*) to select phage and cellular suppressors that define interactions with other proteins; in one approach they will specifically mutagenize candidates like nusA, nusB, and nusE. They will try to isolate nusG ts mutations, and N-resistant nusG mutations, as well as nusG mutations that affect cellular termination. 2) Nun is a protein made by HKO22 that is like Nit binds the same RNA site (named nut) and uses the same cofactors but terminates instead of antiterminating. It works in vitro with purified components, as does N, but slightly defectively, because the RNA does not release. They will look for release, either by the obvious known candidate GreB, or by unknown cellular release factors in extracts. They will isolate release defective mutants of E. coli, under the assumption that arrest of transcription by Nun along with failure of release will induce constipation. They will investigate the structure of Nun and the Nun- RNA complex: the effect of mutants of the nut site known to affect N, RNase protection patterns, the activity of fragments of Nun, the activity of Nun carrying modification of putative critical arginine residues, and (by collaboration) NMR structure of Nun-RNA complex. They will seek a Nun with altered specificity for a mutant nut site and look for suppressors of mutations in accessory factors or RNA polymerase (NusA, NusG, rpoC). They will look for contacts by crosslinking, and characterize protein components of Nun-arrested complexes. They will characterize RNA at sites of arrest (as opposed to recognition). Finally, they will investigate a natural proteolytic cleavage, looking for the protease, and isolate E. coli mutants resistant to the activity of Nun.
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