Real-time monitoring of cell-free translation on a quartz-crystal microbalance.
Real-time monitoring of cell-free translation on a quartz-crystal microbalance.
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DOI：
10.1021/ja9019947
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发表时间：
2009-06
影响因子：
15
通讯作者：
Shuntaro Takahashi;Masaaki Iida;H. Furusawa;Y. Shimizu;T. Ueda;Y. Okahata
中科院分区：
文献类型：
--
作者：
Shuntaro Takahashi;Masaaki Iida;H. Furusawa;Y. Shimizu;T. Ueda;Y. Okahata
蛋白质合成的效率通常受mRNA序列的转录后调控。为了研究蛋白质翻译的反应，我们建立了一个系统，可以使用无细胞翻译混合物和27 MHz石英晶体微天平（QCM）实时监测蛋白质合成。利用编码融合多肽的mRNA，该融合多肽由链霉亲和素结合肽（SBP）标签、一部分蛋白D作为间隔物和SecM捕获序列组成，我们可以跟踪SBP标签在70S核糖体上显示时与链霉亲和素修饰的QCM的结合。因此，我们可以按照质量的变化来跟踪蛋白质合成的单一周转。这种方法使我们能够评估不同抗生素和mRNA序列对不同翻译步骤的影响。根据本研究的结果，我们已经确定，由70S核糖体、mRNA和fMet- trna （fMet）形成起始复合物以及第二氨基酰基- trna进入起始复合物是蛋白质合成的限速步骤。
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.