POLYSOMES & SUBUNITS--STRUCTURE-FUNCTION RELATIONSHIPS

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The objective of this research proposal is to further the understanding of the catalytic role of rRNA in the process of translation. Our goal is to define regions in E. coli rRNA which are involved in particular functions during the different stages of protein synthesis. The primary approach of the laboratory involves mutagenesis of rRNA by in vitro and in vivo methods using a plasmid-borne rrnB operon. Plasmids, particular cell strains and mutagenic techniques have been developed to permit the isolation of lethal as well as nonlethal mutations in sufficient quantities for analysis. Structural and functional features of the mutants will be characterized by a variety of biochemical and genetic systems. Four major topics of translation will be investigated, in each of which rRNA has a central functional role. The four areas include I. Fidelity of translation; functional interactions between tRNA and rRNA. Mutants will be constructed by site-directed mutagenesis or selected for by systems such as TrpE91, following random mutagenesis, to study functional interactions between selected regions of tRNA and rRNA. II. Interactions between mRNA and rRNA at initiation and termination. Translational enhancers will be identified and characterized as will sites of functional interaction between mRNA and rRNA at termination. III. Peptidyl transferase activity in 23S rRNA. Mutations will be constructed at important functional sites involving tRNA and rRNA interactions in domain V of 23S rRNA. IV. The role of 4.5S rRNA, 16S, and 23S rRNA in transport of nascent proteins across the cell membrane. Mutations will be constructed at putative functional sites in 4.5S rRNA and the rRNAs, and effects on transport will be studied. It is anticipated that these studies will contribute to our understanding of the functional role of rRNA by defining in detail structural and functional features of particular regions of the rRNAs and their contributions to the overall process of protein synthesis.