The lack of knowledge on detailed RNA structures has inhibited understanding of the predominant role played by ribosomal RNA (rRNA) in ribosome function. The proposed research focuses on highly conserved regions of rRNA that interct with transfer RNA and small molecule antibiotics. RNA oligonucleotides that correspond to several regions of rRNA will be synthesized, their structures compared to that adopted in intact ribosomes using chemical probing methodology, and their conformations will then be studied in detail using NMR spectroscopy. 16S rRNA contains a highly conserved region of nucleotides near its 3'- terminus (1400 and 1500), called the decoding site, that is involved in both P-site and A-site binding of tRNA anticodon stem-loops. Initial studies on the conformation of this region will focus on smaller (<30 nts) model oligonucleotides; the information derived from these studies will be applied to larger systems comprising several elements of the decoding site. The decoding site of rRNA is also the binding site for aminoglycoside antibiotics that inhibit ribosome function. The interaction of antibiotics, such as neomycin, kanamycin, and hygromycin B, with decoding region oligonucleotides will be characterized by both biochemical methods and NMR. Initial studies suggest that these antibiotics bind to oligonucleotides in the same location as in intact ribosomes. These results encourage the use of model oligonucleotides to understand ribosome-antibiotic interaction. Single-nucleotide mutations in the decoding region lead to antibiotic resistance. The conformations of these oligonucleotides, and their complexes with antibiotics will also be probed by NMR, and the results compared to that for wild-type sequence. Hopefully, these studies will determine the structural origin of antibiotic resistance for this class of drugs. The 3'-CCA end of tRNAs bound in the ribosomal P-site interact with a universally conserved hairpin loop, consisting of 3 guanines. Mutation of these nucleotides disrupts tRNA interaction. The conformation of this loop will be determined using NMR methods. Mutant and wild-type sequences will be compared and the role of a specific modified ribose residue will be investigated. In addition, the interaction of these loops with simple oligonucleotides containing the tRNA CCA sequence will be studied to determine the capability of these loops to interact with this portion of tRNA. The proposed research addresses the general question of RNA structure and RNA-ligand interaction within the context of ribosomal function. An understanding of tRNA recognition by the ribosome would reveal one of the essential interactions in biology. The proposed research will attempt to characterize the structural mode of action of a class of antibiotics, the aminoglycosides. The current crisis in antibiotic resistance demands a more detailed understanding of the molecular mechanisms of antibiotic action.
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