Key result
Real-time monitoring using a quartz-crystal microbalance determined that initiation complex formation and accommodation of the second aminoacyl-tRNA are rate-limiting steps in protein synthesis.
A novel QCM-based system allows real-time monitoring of cell-free protein synthesis, revealing that initiation complex formation and second aminoacyl-tRNA accommodation are rate-limiting steps.
No immediate clinical implications; leaves open whether targeting these steps optimizes cell-free protein synthesis in research models.
The efficiency of protein synthesis is often regulated post-transcriptionally by sequences within the mRNA. To investigate the reactions of protein translation, we established a system that allowed real-time monitoring of protein synthesis using a cell-free translation mixture and a 27 MHz quartz-crystal microbalance (QCM). Using an mRNA that encoded a fusion polypeptide comprising the streptavidin-binding peptide (SBP) tag, a portion of Protein D as a spacer, and the SecM arrest sequence, we could follow the binding of the SBP tag, while it was displayed on the 70S ribosome, to a streptavidin-modified QCM over time. Thus, we could follow a single turnover of protein synthesis as a change in mass. This approach allowed us to evaluate the effects of different antibiotics and mRNA sequences on the different steps of translation. From the results of this study, we have determined that both the formation of the initiation complex from the 70S ribosome, mRNA, and fMet-tRNA(fMet) and the accommodation of the second aminoacyl-tRNA to the initiation complex are rate-limiting steps in protein synthesis.
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Takahashi et al. (2009) studied this question. Real-time monitoring using a 27 MHz quartz-crystal microbalance was evaluated on Real-time monitoring of protein synthesis as a change in mass. Real-time monitoring using a quartz-crystal microbalance determined that initiation complex formation and accommodation of the second aminoacyl-tRNA are rate-limiting steps in protein synthesis.
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