This project will make use of photoaffinity labeling to characterize the binding sites for tRNA on E. coli ribosomes, and in vitro mutagenesis will be employed to study structure-function relationships in ribosomal RNA and ribosomal proteins. (1) Photolabile azidopurines will be introduced into the D,T and anticodon loops, and the anticodon stem, of tRNA molecules using chemical and enzymatic reconstruction techniques. Labeled, aminoacylated tRNAs will be bound to both P and A sites of 70S ribosomes and irradiated to induce cross-linking. Fragments encompassing the anticodon arm of tRNA Phe will be covalently attached to 30S subunits in a similar fashion. Ribosomal components cross-linked to tRNA will be identified and the sites of reaction determined. These experiments are expected to aid in defining the topography of tRNA binding sites on the ribosome. (2) The photochemistry of the azidouprines will be investigated under a variety of conditions in order to better understand the mechanism of cross-linking and the structure of the cross-linked products. The formation and structure of a covalent, site-specific complex between 8-azidoadenosine and RNase A will be studied by conventional and rapid-scanning x-ray crystallography in the hope of identifying reaction intermediates. (3) Site-directed alterations will be introduced into various segments of the genes for E. coli 16S and 23S rRNAs using in vitro mutagenesis. Particular attention will be focused upon the properties of three lethal mutations and their intragenic suppressor, all of which occur within two phylogenetically conserved sequences at the 3' end of 16S rRNA. In addition, the physiological consequences of mutations within the binding site for protein S8 will be examined. Alterations will also be introduced at other functional sites in 16S and 23S rRNAs. Effects of the variant rRNAs on cell growth. rRNA, processing, subunit assembly and protein synthesis will be assessed. The results will lead to a more precise understanding of rRNA function. (4) Site-specific mutagenesis will be employed to investigate the functional domains of ribosomal proteins. Targets will be selected on the basis of sequence comparisons and other available data. Efforts will focus on establishing the stability and RNA-binding properties of mutant variants of S8, a protein that plays a critical role in both 30S subunit assembly and translational regulation.
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