The goal of this proposal is to understand the relationship between the structure of tRNA and its function in aminoacylation and protein synthesis. We have developed efficient enzymatic procedures to cleave tRNA in the anticodon loop, remove one or more nucleotides, insert other nucleotides and reseal the tRNA to produce a variant molecule with a defined chemical change in the anticodon. Similar procedures will be developed to alter other parts of the tRNA molecule. The substrate sepcificity of the enzymes used in this procedure are sufficiently broad that a wide variety of base and sugar modifications can be inserted into the polynucleotide chain. In addition, an in vitro transcription procedure will be developed to obtain unmodified but fully processed tRNAs from tRNA gene. Taken together, these methods give a high degree of synthetic control over a crucial molecule in the translation mechanism. The methods should be applicable to other RNA molecules. We will use this technology to prepare variant tRNAs to examine several aspects of tRNA function. First, we want to evaluate the relative role of different regions of tRNA in the specificity of its interaction with its cognate tRNA synthetase. The kinetics of aminoacylation of tRNAs with modifications in the anticodon, the D stem region and the acceptor stem region will be compared. Second, the codon-anticodon interaction of tRNAPhe will be examined on poly U programmed E. coli ribosomes. Steady state and single turnover kinetics of phenylalanine tRNAs modified in the anticodon loop will be determined.
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