Translation, in vivo protein synthesis, is a vital cellular process to produce enzymes that perform almost every critical function in the cell. These functions include gene transcription, gene repair, protein synthesis, and protein folding/degradation. Therefore, understanding translation is essential in controlling cell function. The ribosome selects correct transfer RNA (tRNA) based on mRNA(codon)-tRNA(anticodon) interaction to synthesize proteins with correct sequences. The selection is composed of two sub-steps - initial selection and proofreading. The purpose of candidate's proposed research is to elucidate the mechanism of the initial selection. During the initial selection, ternary complex of elongation factor Tu (EF-Tu), tRNA, and GTP delivers tRNA to the ribosome. Only when codon matches with anticodon, EF-Tu hydrolyzes GTP and changes its conformation to dissociate from the ribosome. Candidate hypothesizes that the codondependent GTP hydrolysis on EF-Tu is energized by tRNA motion, which is induced by the ribosomal conformational change. The conformational change of the ribosome upon the binding of the ternary complex depends on codon-anticodon interaction. Therefore, tRNA acts as a communication channel between the ribosome and EF-Tu. To test the hypothesis, candidate proposes to monitor individual working ribosome in real-time through single molecule fluorescence techniques. Single molecule methods enable high time-resolution real-time monitoring of individual steps in non-synchronizable multi-step enzymatic processes. Candidate proposes following specific aims to test the hypothesis: #1 Construct an experimental system to monitor tRNA motion, elongation factor Tu (EF-Tu) movement, and GTP hydrolysis event through single molecule fluorescence techniques 1) Achieve 3 ms time resolution to monitor fluorescence intensity changes, 2) Fluorescently label EF-Tu without disturbing its internal sequence, and 3) test fluorescent GTP analogues to monitor EF-Tu movement and GTP hydrolysis simultaneously, #2 Achieve the highest possible signal to noise ratio (S/N) for single molecule fluorescence resonance energy transfer measurements 1) Optimize instruments for highest possible S/N, 2) Optimize oxygen scavenger system, 3) Improve data analysis algorithm based on hidden Markov models, and 4) Setup a single photon counting system, #3 Relate tRNA motion to GTP hydrolysis and EF-Tu dissociation 1) Monitor GTP hydrolysis and tRNA motion simultaneously, and 2) Monitor EF-Tu movement and tRNA motion simultaneously. Successful completion of proposed research will enhance our understandings about how the translation machinery achieves high accuracy protein synthesis.
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