The objective of this project is to study the biochemical properties of tRNA molecules prepared by in vitro transcription that have defined structural modifications at various positions in the polynucleotide chain. These experiments will permit a better understanding of how tRNA is recognized by the enzymes that interact with it. It is possible that certain mutant tRNAs will block an enzyme reaction pathway at a new step, revealing more about the mechanism of the enzyme. Finally, since the structure of the mutant RNAs will be carefully studied, more will be learned about the folding of RNAs into a tRNA-like structure. Mutant E. coli tRNAs will be assayed on E. coli ribosomes using several equilibrium and kinetic assays for binding, dipeptide formation and proofreading steps. tRNA nucleotides involved in specific ribosome tRNA contacts will be identified. The nucleotides important for the recognition of yeast and E. coli tRNA Phe by their cognate synthetases will be compared. The available co-crystal structure of the E. coli glutamine tRNA-enzyme complex will be used to design experiments to understand a conformational isomerization that appears to occur in the reaction pathway. RNAs which are expected to fold like tRNA but have sequences very different from tRNA will be designed and studied. The structure and activity of several isolated tRNA domains will be studied.
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