This proposal will continue to develop enzymatic and chemical techniques for the synthesis and modification of RNA molecules and apply them to three problems involving the structure and function of RNA. A series of oligoribonucleotides will be made which duplicate known secondary structural features in RNA molecules. These include a number of short helices with different sequences, """"""""dangling"""""""" terminal nucleotides, mismatches and hairpin loops. The helix-coil transition of each oligomer will be measured and the data will be analyzed to obtain the thermodynamic parameters of the reaction. These data will be used to improve the empirical parameters for the prediction of RNA secondary structure. The specific binding of R17 coat protein to its translational regulation site on R17 RNA will be studied in greater detail. Chemical modification of the RNA will be used to identify contacts between the two. Fluorescence methods will be used to study the thermodynamics of the binding. Varients of the binding sequence will be synthesized to better understand the specificity of the interaction. The effect of mRNA sequence and structure on the specificity and strength of translational initiation will be studied. Varients of the R17 replicase initiation regions will be synthesized and their ability to form an initiation complex and promote dipeptide synthesis will be assayed. The sequence 3' end of E. coli 16S rRNA will be altered to study its effect in translational initiation.
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