Translation, in vivo protein synthesis, is vital process in maintaining cell life by producing enzymes performing almost every critical function in the cell including gene transcription, gene repair, protein synthesis, and protein folding/degradation. Understanding translation is essential in controlling cell function/life by offering ways to control enzyme production in the cell. The ribosome selects the correct transfer RNA (tRNA) based on mRNA(codon)-tRNA(anticodon) interaction to synthesize protein with the correct sequence. The selection is composed of two sub-steps - initial selection and proofreading. Candidate is currently trying to elucidate the mechanism of proofreading. During the initial selection, ternary complex of elongation factor Tu (EF-Tu), tRNA, and GTP delivers tRNA to the mRNA/ribosome complex. Only when codon matches with anticodon, EF-Tu hydrolyzes GTP and changes conformation to dissociate from the ribosome. Candidate hypothesizes that this recognition process (codon-dependent GTP hydrolysis on EF-Tu) is enabled by tRNA fluctuations, dynamics of which is determined by codon-anticodon interaction. Therefore, tRNA acts as a communication channel between the ribosome decoding site and EF-Tu. To examine the hypothesis, candidate proposes to monitor individual working ribosome in real-time through single molecule fluorescence measurement. Single molecule measurement enables 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 movement, elongation factor Tu (EF-Tu) movement, and GTP hydrolysis through single molecule fluorescence resonance energy transfer (SM FRET) 1) Achieve 3 ms time resolution to monitor the dynamics, 2) Label EF-Tu and test fluorescent GTP analogues to monitor EF-Tu movement and GTP hydrolysis, #2 Achieve the highest possible signal to noise ratio (S/N) forSM FRET measurements 1) Optimize instrumentation for highest possible S/N, 2) Optimize oxygen scavenger system, 3) Implement noise removal algorithm based on stochastic prediction, #3 Relate tRNA motion to GTP hydrolysis and EF-Tu dissociation 1) Monitor GTP hydrolysis and tRNA motion simultaneously, 2) Monitor EF-Tu movement and tRNA motion simultaneously. Successful completion of proposed research will greatly enhance our understandings in translation. Understanding how the translation machinery synthesizes proteins with such an unusually high accuracy will open ways to control cell function/life.
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