In this project, affinity labeling will be used to characterize the sites of interaction of tRNA with E. coli ribosome, and recombinant DNA techniques will be employed to study the function of ribosomal components altered by specifically targeted mutations. (1) Photolabile probes will be introduced into tRNA molecules by derivatization of specific bases with aryl azide reagents, or by the replacement of purine residues with 8-azidopurines. Labeled, aminoacylated tRNAs will be bound to both P and A sites of 70S ribosomes and irradiated to induce cross-linking. Ribosomal components covalently attached to tRNA will be identified and the sites of insertion determined. In addition, the structure of complexes resulting from the reaction of Epsilon-bromoacetyl-Lys-tRNA-Lys with 50S subunit proteins and 23S RNA at the ribosomal P site will be defined. These experiments are expected to aid in delineating the topography of the tRNA binding sites on the ribosome. (2) Site-specific alterations will be introduced into various segments of the gene for E. coli 16S RNA with the aid of bisulfite and oligonucleotide-directed mutagenesis in vitro. One series of mutations will be used to probe an evolutionarily conserved sequence near the 3' end of the RNA that is adjacent to the tRNA anticodon at the ribosomal decoding site. A second set, in the binding site for protein S8, will be utilized to assess the structural features of the RNA that are required for protein recognition and interaction. The mutagenized segments will be cloned into plasmid-encoded ribosomal RNA operons in place of the corresponding wild-type sequences and used to transform an appropriate E. coli host. Effects of the variant 16S rRNA molecules on properties such as cell growth, RNA processing, ribosome assembly, and translational fidelity will be determined. Mutant transcripts will also be synthesized in vitro for the analysis of protein-RNA association. The results will lead to a more precise understanding of structure-function relationships in ribosomal RNA. (3) Site-specific mutagenesis will also be employed to investigate the functional domains of ribosomal proteins. Target sites will be selected on the basis of sequence comparisons and other available data. Initial efforts will focus on protein S8, which plays a critical role in both 30S subunit assembly and translational regulation and therefore exhibits the property of mutifunctionality that is typical of many ribosomal proteins.
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