The principle activity for which this application seeks support is determination of the structures of physiologically important RNAs by NMR and X-ray crystallography. The ultimate objective is a chemical understanding of the biological properties of the RNAs studied. Since RNAs play critical roles in gene expression, the knowledge sought is fundamental to our understanding of processes vital to all organisms, including humans. Two specific problems are to be investigated: the regulation of ribosomal protein synthesis in bacteria, and the pseudouridylation of ribosomal RNA transcripts in eukaryotes. In bacteria, ribosomal protein synthesis is feed-back regulated at the translational level by mechanisms that depend on interactions between specific ribosomal proteins and sequences within the mRNAs that encode them, many of which are polycistronic. The two such systems of immediate concern are the spc operon/S8 system, and L10 operon./L10 system, but time permitting, others will be investigates; e.g. the alpha operon/L4 system. The mRNA sequence critical for the regulation of each ribosomal protein operon will be determined as precisely as possible. Structures will be obtained of the complexes that form between these RNA sequences and the ribosomal proteins that bind to them. Hypotheses about the mechanism of translational repression, formulated on the basis of those structures, will then be tested biochemically. Since bacteria regulate ribosomal protein synthesis by mechanisms unlike those used by eukaryotes, it is conceivable that new ways of specifically inhibiting bacterial growth will be revealed. The second problem to be investigated is posed by the boxH/ACA snoRNP system responsible for pseudouridylating rRNA transcripts in higher organisms. The RNA components of these snRNPs are postulated to interact with sequences in rRNA transcripts in a way that has no precedent in the literature. Biochemical experiments will be done to determine whether RNA/RNA interactions of the sort hypothesized are possible, and structures will be obtained for a snoRNA, with and without an appropriate rRNA sequence bound. ? ?
