Structure and Mechanism of Class II tRNA Synthetases

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DESCRIPTION (provided by applicant): Aminoacyl- tRNA synthetases are essential in the decoding of genetic information in all living systems. Despite their relatively early discovery and recent extensive structural characterization, significant questions concerning the details of their mechanisms and molecular functions remain to be addressed. During the previously funded cycle, the principal investigator developed a fairly complete picture of the adenylation reaction catalyzed by histididyl-tRNA synthetase using crystallography and molecular genetics, identifying mobile regions of the enzyme important for catalysis. He further showed that tRNA binds to the enzyme in multiple steps, and that residues within the motif 2 loop are important for discriminator base specificity. Other studies investigated evolutionary aspects of the HisRS family, including the discovery of a new family of HisRS-like proteins with a role in the regulation of histidine biosynthesis. He also provided the first evidence for editing in ThrRS. A number of important questions related to tRNA synthetase function remain to be resolved, including the role of structural changes in both the enzyme and the tRNA during the reaction. In this project renewal, the principal investigator will investigate the dynamics of tRNA synthetase:tRNA interactions in the histidine and threonine systems by use of fluorescence assays to monitor individual steps in the adenylation and aminoacylation reactions. Specific aims are to: 1) define the role of enzyme conformational dynamics in the adenylation reaction in two class aminoacyl-tRNA synthetases by transient kinetic analysis; 2) determine the rate of the aminoacylation reaction by transient kinetic analysis and investigate whether there are significant intermediates in the tRNA binding process; 3) investigate the basis of tRNA editing in ThrRS by using transient kinetic analysis to establish the quantitative basis of amino acid selection and the rates of threonylation and serylation in wild type and mutant enzymes. Through correlation of fluorescence changes to mechanistic steps in the aminoacylation reaction catalyzed by HisRS and ThrRS, the principal investigator hopes to gain insight into the fundamental question of how enzymes that recognize nucleic acids in a sequence specific fashion utilize binding-induced conformational changes to promote catalysis.