Salas-Marco Joe, Bedwell David M
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294-2170, USA.
J Mol Biol. 2005 May 13;348(4):801-15. doi: 10.1016/j.jmb.2005.03.025.
The suppression of stop codons (termed translational readthrough) can be caused by a decreased accuracy of translation elongation or a reduced efficiency of translation termination. In previous studies, the inability to determine the extent to which each of these distinct processes contributes to a readthrough phenotype has limited our ability to evaluate how defects in the translational machinery influence the overall termination process. Here, we describe the combined use of misincorporation and readthrough reporter systems to determine which of these mechanisms contributes to translational readthrough in Saccharomyces cerevisiae. The misincorporation reporter system was generated by introducing a series of near-cognate mutations into functionally important residues in the firefly luciferase gene. These constructs allowed us to monitor the incidence of elongation errors by monitoring the level of firefly luciferase activity from a mutant allele inactivated by a single missense mutation. In this system, an increase in luciferase activity should reflect an increased level of misincorporation of the wild-type amino acid that provides an estimate of the overall fidelity of translation elongation. Surprisingly, we found that growth in the presence of paromomycin stimulated luciferase activity for only a small subset of the mutant proteins examined. This suggests that the ability of this aminoglycoside to induce elongation errors is limited to a subset of near-cognate mismatches. We also found that a similar bias in near-cognate misreading could be induced by the expression of a mutant form of ribosomal protein (r-protein) S9B or by depletion of r-protein L12. We used this misincorporation reporter in conjunction with a readthrough reporter system to show that alterations at different regions of the ribosome influence elongation fidelity and termination efficiency to different extents.
终止密码子的抑制作用（称为翻译通读）可能是由翻译延伸准确性的降低或翻译终止效率的降低引起的。在先前的研究中，无法确定这些不同过程各自对通读表型的贡献程度，这限制了我们评估翻译机制中的缺陷如何影响整体终止过程的能力。在这里，我们描述了错配和通读报告系统的联合使用，以确定这些机制中的哪一种导致了酿酒酵母中的翻译通读。错配报告系统是通过将一系列近同源突变引入萤火虫荧光素酶基因中功能重要的残基而产生的。这些构建体使我们能够通过监测由单个错义突变失活的突变等位基因的萤火虫荧光素酶活性水平来监测延伸错误的发生率。在这个系统中，荧光素酶活性的增加应该反映野生型氨基酸错配水平的增加，这提供了翻译延伸整体保真度的估计。令人惊讶的是，我们发现巴龙霉素存在下的生长仅刺激了所检测的一小部分突变蛋白的荧光素酶活性。这表明这种氨基糖苷诱导延伸错误的能力仅限于近同源错配的一个子集。我们还发现，核糖体蛋白（r-蛋白）S9B的突变形式的表达或r-蛋白L12的缺失可诱导近同源错读中的类似偏差。我们将这种错配报告系统与通读报告系统结合使用，以表明核糖体不同区域的改变对延伸保真度和终止效率的影响程度不同。
