This project will utilize photoaffinity labeling techniques to characterize the topography of tRNA binding sites on the E. coli ribosome, and in vitro mutagenesis to study structure-function relationships in ribosomal proteins and rRNA. (1) Photoreactive nucleosides will be introduced into tRNA molecules by chemical and enzymatic technique or by in vitro transcription. New reconstruction methods will allow us to place the modified bases at any desired position. The tRNA derivatives will be cross-linked tot he ribosome and the labeled components will be identified. Particular emphasis will be placed on determining the orientation of tRNA in the A,P and E sites, the identity of cross-linked residues in both rRNA and ribosomal proteins, and the nature of any changes that occur in the relative positions of tRNA and ribosome during peptidyl transfer and translocation. (2) New photolabile nucleosides will be prepared and their photochemistry assessed. In addition, suitably protected forms of photoreactive nucleoside monomers will be prepared for use in the automated, solid- phase synthesis of oligodeoxyribonucleotides. These synthons should be useful not only for the preparation of photoreactive tRNA derivatives, but also for antisense, catalytic or other RNAs where the proteins will be studied with the aid of site-directed mutagenesis in conjunction wit biochemical analysis. In particular, the structural features of protein S8 that are required for its specific interactions with 16S rRNA and spc operon mRNA will be further defined. Moreover, the role of L27 in 50S- subunit function will be investigated using a genetic system in which the activity of the ribosomes is entirely dependent upon the assembly of mutant protein into 50S subunits. As L27 is a target for a number of macrolide antibiotics, this work may shed new light on the structural aspects of antibiotic resistance. (4) Site-directed alterations will be introduced into several segments of the genes for 16S and 23S rRNAs using in vitro mutagenesis. The subjects of investigation include three lethal point mutations and their intragenic suppressor, all of which occur within a highly conserved sequence at the 3' end of 16S rRNA. The effects of mutations at two sites within the 23S rRNA will also be examined, the conserved G1945 region adjoining the 3' end of the P-site tRNA and the sarcin stem-loop which may mediate the influence of elongation factors upon 50S subunit structure.
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