The goal of the proposed research is to understand the mechanisms by which ribosomal proteins recognize specific RNA sites, and the influence protein binding may have on RNA structure. Thermodynamic, chemical modification, and genetic techniques will be used to study three protein-RNA interaction systems. Using an in vitro transcription system, sets of rRNA or mRNA fragments differing in length or sequence will be prepared, and their ability to specifically bind proteins tested. Physical studies of the RNA fragments and the RNA-protein complexes will show whether protein binding influences the RNA conformation. The three systems to be studied are: i) an extended 97 base RNA hairpin containing the recognition site for protein S15. This RNA is already known to contain unusually high affinity sites for ethidium intercalation, and to undergo a distinct conformational change upon ethidium binding. Whether protein binding is coupled to this conformational change will be investigated. ii) The protein S4 recognizes both the 16S rRNA, and the mRNA coding for S4 and several other ribosommal proteins. Regions of the mRNA required for S4 binding have already been defined. Further work will delineate the specific nucleotides required for recognition in each RNA, and compare the thermodynamics of binding to each RNA. Genetic systems capable of selecting mutants in the rRNA, mRNA, or protein defective in recognition will be used to help detect important recognition features. iii) The proteins L10 and L11 bind cooperatively to a limited, about 100 base region of the 23S rRNA. This region of rRNA is highly conserved and important for the GTPase activity of translating ribosomes. The protein binding sites, mechanism of cooperatively, and possibility of reconstituting GTPase activity in a small complex of proteins and rRNA will be investigated.
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