RNA-BINDING SITE OF A TRANSLATIONAL REPRESSOR

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It is increasingly appreciated that the recognition of specific RNA sequences by proteins is a fundamental aspect of a variety of normal cellular processes. Additional attention has been focussed on this problem because of the critical roles played by RNA-protein interactions in the life cycle of Human Immunodeficiency Virus, the cause of AIDS. The long term goal of this work is to contribute to a general understanding of the fundamental nature of RNA-protein interactions by understanding one particular model of such an interaction in detail. The coat protein of the bacterial virus MS2 is a genetic regulatory protein. It binds a specific RNA stem-loop (the translational operator) to repress translation of the viral replicase gene. Previous work in this laboratory has defined the RNA binding site through the isolation of coat protein mutants which either have reduced affinity for RNA, have increased affinity for RNA, or show altered RNA binding specificity. The main objective of the present proposal is to combine our separate knowledge of the structures of the coat protein RNA-binding site and its RNA ligand into a picture of how the two molecules interact. Two general approaches are proposed. First, since the mutant proteins must exert their effects by either removing contacts or adding new ones, the specific nature of these missing or added contacts can be probed by asking how mutant proteins differ from wild-type in their abilities to accommodate chemical modification or nucleotide substitution at specific sites in the RNA. Second, evolutionary variants of the MS2 virus utilize homologous coat proteins to bind divergent RNA structures. It is proposed to define the protein structural determinants of RNA binding specificity using a combination of genetic and biochemical methods.