Dynamics in Translation: the Role of Fluctuation in Protein Synthesis

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date7/1/086/30/09

Funding

  • National Institute of General Medical Sciences: $242,238.00

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