STRUCTURE/FUNCTION OF SRP RNA

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The objective of this research proposal is to advance the 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 secretion. A major component of all SRPs is an RNA molecule, regions of which are universally conserved. Our goal 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 particular aspects of SRP-mediated protein secretion and SRP assembly by using a combination of site-directed mutagenesis, site- directed cross-linking, and phylogenetic comparisons with cloned SRPcomponents. The major topics to be investigated in this proposal are: (1) The molecular mechanisms involved in signal recognition and protein translocation. SRPs reconstituted with mutant SRP RNAs will be used to test the translocation competence of secretory proteins in a cell-free translation-translocation system. The individual steps which are affected in the SRP-cycle, such as signal recognition, translation arrest, and release of the arrest, will be identified. (2) SRP assembly. Mutants of SRP RNA will be constructed by systematic site-directed mutagenesis, or selected after random mutagenesis, to study the interactions of the SRP proteins with the SRP RNA. Information about the binding sites on the proteins will be obtained by testing the ability of mutant polypeptides to interact with the SRP RNA or with partially assembled ribonucleoprotein particles (RNPs). Photochemical cross-linking with modified nucleotides introduced into the SRP RNA at predetermined positions will be used to identify neighborhoods at the single nucleotide and amino acid level. (3) Biophysical characterization of an RNA tetranucleotide loop interaction with protein SRP19. These studies will focus on the specific features of a critical protein-RNA interaction in a conserved region of the SRP and define its relationship to the overall process of protein translocation.