TRNA RECOGNITION AND CATALYSIS BY E COLI HISRS

University Of Vermont &St Agric College, Burlington VT

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Abstract

The objective of the proposed work is to understand how aaRS utilize sequence-specific RNA recognition for optimal catalytic rate and specificity. Several recent reports suggest that aaRS have regulatory functions in control over cell growth and division that were not previously noticed, and which may be relevant to the cancer problem. In one report, reduced HisRS or LysRS activity was associated with G1 arrest due to a reduction in cyclin D1 expression but not cyclin E expression. A clearer understanding of how these enzymes function at the molecular level may eventually lead to the development of cancer intervention strategies. The specific aims of the project are to 1) determine which amino acid side chains interact with the tRNA and how individual mutations affect the overall rate of the aminoacylation reaction, 2) derive a free energy profile for the reaction through determination of individual rate constants for the aminoacylation reaction, and 3) quantitate the extent of coupling between tRNA recognition and aminoacylation, particularly among contacts to the acceptor arm and the orientation of the A76 acceptor nucleotide. These aims will be carried out through the use of presteady state kinetics methods including stopped-flow fluorimetry and quenched-flow trapping experiments.