Structure and function of SRP RNA

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DESCRIPTION (provided by applicant): The objective of this research proposal is to advance the molecular understanding of the basic process of protein transport across biological membranes. Our particular focus is on the signal recognition particle (SRP) as it plays a central role in protein targeting and secretion. The SRP RNA is an essential component of every SRP and contains several conserved features of functional importance. The goal of our research is to understand the structural and functional role of this RNA in the human system. We will define those regions of the human SRP RNA which are involved in SRP-mediated protein secretion and SRP assembly by using a combination of site-directed mutagenesis, site-directed cross-linking, structure probing, comparative analyses, biochemical, and biophysical studies. The major topics to be investigated are: (1) Mechanisms that involve SRP RNA in SRP assembly, signal recognition, and protein translocation. Complexes will be reconstituted from recombinant components to study the formation of the SRP and translocation competence of secretory proteins. The effects of altered complexes on individual events in the SRP-cycle, such as signal peptide recognition, translation arrest, and release of the arrest, will be identified. (2) Structural and functional role of proteins SRP68 and SRP72 within the human SRP. The interactions of SRP RNA with SRP68 and SRP72 will be studied by systematic site-directed mutagenesis. RNA-protein neighborhoods will be determined by using site-directed photochemical cross-linking followed by the identification of the cross-linked nucleotides and amino acids. The data from these experiments will be viewed collectively and used to generate a three-dimensional molecular model of human SRP. (3) Biophysical characterization of the M-domain of SRP54 which binds to the SRP RNA helix 8 and the signal as well as of recombinant signal fusion protein derivatives (SFPs) manipulated to contain altered signal peptides. These studies will focus on the specific features of two critical interactions and define their dynamic relationship to SRP function.