Luciferase-based reporter system for in vitro evaluation of elongation rate and processivity of ribosomes.
Luciferase-based reporter system for in vitro evaluation of elongation rate and processivity of ribosomes.
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DOI：
10.1093/nar/gkab121
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发表时间：
2021-06-04
影响因子：
14.9
通讯作者：
Tamm T
中科院分区：
文献类型：
--
作者：
Kisly I;Kattel C;Remme J;Tamm T
翻译的延伸步骤对蛋白质的丰度、折叠和质量以及mRNA的稳定性起着关键的作用。然而，对翻译伸长的控制还没有得到彻底的调查。在这项研究中，我们进一步开发了一个Renilla-萤火虫荧光素酶融合报告系统，以研究核糖体的体外伸长率和加工能力，而不依赖于起始和终止步骤。构建的报告基因包含一个编码框内Renilla荧光素酶、特定结构域部分和萤火虫荧光素酶的ORF。这样的报告结构使得能够对特定结构域的合成进行定量和单独的评估。作为原理的证明，分析了三个不同长度和结构的蛋白质结构域的合成。使用无细胞翻译实验，核糖体的延伸率和加工性都随着合成结构域的不同而变化。此外，由10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率和加工性。所有这些结果都与已知的体内伸长动力学一致。总体而言，本报告提出的方法为研究有助于延长翻译步骤的各方面提供了一个框架。
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.
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影响因子：
9.8
作者：
Charneski CA;Hurst LD
通讯作者：
Hurst LD
影响因子：
7.2
作者：
Dever TE;Dinman JD;Green R
通讯作者：
Green R
DOI：
10.1186/1748-7188-6-26
发表时间：
2011-11-24
期刊：
Algorithms for molecular biology : AMB
影响因子：
--
作者：
Lorenz R;Bernhart SH;Höner Zu Siederdissen C;Tafer H;Flamm C;Stadler PF;Hofacker IL
通讯作者：
Hofacker IL
影响因子：
5.3
作者：
IIZUKA, N;NAJITA, L;SARNOW, P
通讯作者：
SARNOW, P
影响因子：
5.4
作者：
Brasey, A;Lopez-Lastra, M;Sonenberg, N
通讯作者：
Sonenberg, N