Institute of Molecular and Cell Biology, University of Tartu, Tartu 51010, Estonia.
Nucleic Acids Res. 2021 Jun 4;49(10):e59. doi: 10.1093/nar/gkab121.
The elongation step of translation is a key contributor to the abundance, folding and quality of proteins and to the stability of mRNA. However, control over translation elongation has not been thoroughly investigated. In this study, a Renilla-firefly luciferase fusion reporter system was further developed to investigate the in vitro elongation rate and processivity of ribosomes independent of the initiation and termination steps. The reporter mRNA was constructed to contain a single ORF encoding in-frame Renilla luciferase, a specific domain moiety and firefly luciferase. Such a reporter structure enables the quantitative and individual evaluation of the synthesis of a specific domain. As a proof of principle, the synthesis of three protein domains of different lengths and structures was analyzed. Using a cell-free translation assay, both the elongation rate and processivity of ribosomes were shown to vary depending on the domain synthesized. Additionally, a stalling sequence consisting of ten rare arginine codons notably reduced the elongation rate and the processivity of the ribosomes. All these results are consistent with the previously known dynamics of elongation in vivo. Overall, the methodology presented in this report provides a framework for studying aspects that contribute to the elongation step of translation.
翻译延伸步骤是蛋白质丰度、折叠和质量以及 mRNA 稳定性的关键贡献因素。然而，对翻译延伸的控制还没有被彻底研究。在这项研究中，我们进一步开发了一种海肾荧光素酶-萤火虫荧光素酶融合报告系统，用于在不考虑起始和终止步骤的情况下，体外研究核糖体的延伸速度和延伸的连贯性。报告基因 mRNA 被构建为包含一个单一的 ORF，该 ORF 编码框架内的海肾荧光素酶、一个特定的结构域部分和萤火虫荧光素酶。这种报告基因结构可以定量和单独评估特定结构域的合成。作为原理的证明，我们分析了三种不同长度和结构的蛋白质结构域的合成。使用无细胞翻译测定法，核糖体的延伸速度和延伸的连贯性都显示出取决于所合成的结构域而变化。此外，由十个稀有精氨酸密码子组成的停滞序列显著降低了核糖体的延伸速度和延伸的连贯性。所有这些结果都与体内已知的延伸动力学一致。总的来说，本报告中提出的方法为研究翻译延伸步骤的各个方面提供了一个框架。
