Department of Biomolecular Engineering, Tokyo Institute of Technology, B-53, 4259 Nagatsuta, Yokohama 226-8501, Japan.
J Am Chem Soc. 2012 Apr 18;134(15):6793-800. doi: 10.1021/ja300993d. Epub 2012 Apr 10.
During translation, the biosynthesis of polypeptides is dynamically regulated. The translation rate along messenger RNA (mRNA), which is dependent on the codon, structure, and sequence, is not always constant. However, methods for measuring the duration required for polypeptide elongation on an mRNA of interest have not been developed. In this work, we used a quartz crystal microbalance (QCM) technique to monitor mRNA translation in an Escherichia coli cell-free translation system in real time. This method permitted us to evaluate the translation of proteins of interest fused upstream of a streptavidin-binding peptide (SBP) fusion protein. The translation of mRNA encoding the SBP fusion protein alone was observed as a mass increase on a streptavidin-modified QCM plate. Addition of the protein of interest resulted in a delay in the mass change corresponding to the traveling time of the ribosome along the coding region of the protein of interest. With this technique, the lengths of coding sequences, codon usages, influences of unique sequences, and various protein-coding sequences were evaluated. The results showed that the traveling time of the translating ribosome depends on the length of the coding region translated but is also affected by the sequence itself. Differences in the time lags for various proteins imply that mRNA coding sequences may regulate gene expression.
在翻译过程中，多肽的生物合成是动态调节的。沿信使 RNA（mRNA）的翻译速度取决于密码子、结构和序列，并不总是恒定的。然而，尚未开发出用于测量在感兴趣的 mRNA 上多肽延伸所需的持续时间的方法。在这项工作中，我们使用石英晶体微天平（QCM）技术实时监测大肠杆菌无细胞翻译系统中的 mRNA 翻译。该方法允许我们评估融合在上游的生物素结合肽（SBP）融合蛋白的目的蛋白的翻译。单独编码 SBP 融合蛋白的 mRNA 的翻译表现为在链霉亲和素修饰的 QCM 板上的质量增加。添加目的蛋白会导致与核糖体沿目的蛋白的编码区行进的时间相对应的质量变化延迟。使用该技术，可以评估编码序列的长度、密码子使用情况、独特序列的影响以及各种蛋白质编码序列。结果表明，翻译核糖体的行进时间取决于翻译的编码区的长度，但也受到序列本身的影响。各种蛋白质的时间滞后差异意味着 mRNA 编码序列可能调节基因表达。
