Ribosomal pauses are a critical part of cotranslational events including protein folding and localization. However, extended ribosome pauses can lead to ribosome collisions, resulting in the activation of ribosome rescue pathways and turnover of protein and mRNA. While this relationship has been known, there has been little exploration of how ribosomal stalls impact translation duration at a quantitative level. We have taken a method used to measure elongation time and adapted it for use in Saccharomyces cerevisiae to quantify the impact of elongation stalls. We find, in transcripts containing Arg CGA codon repeat-induced stalls, a Hel2-mediated dose-dependent decrease in protein expression and mRNA level and an elongation delay on the order of minutes. In transcripts that contain synonymous substitutions to nonoptimal Leu codons, there is a decrease in protein and mRNA levels, as well as similar elongation delay, but this occurs through a non-Hel2-mediated mechanism. Finally, we find that Dhh1 selectively increases protein expression, mRNA level, and elongation rate. This indicates that distinct poorly translated mRNAs will activate different rescue pathways despite similar elongation stall durations. Taken together, these results provide new quantitative mechanistic insight into the surveillance of translation and the roles of Hel2 and Dhh1 in mediating ribosome pausing events.

中文翻译：

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酵母中延伸停滞的量化及其对基因表达的影响

核糖体暂停是包括蛋白质折叠和定位在内的共翻译事件的关键部分。然而，延长的核糖体暂停会导致核糖体碰撞，从而导致核糖体救援途径的激活以及蛋白质和 mRNA 的周转。虽然这种关系是已知的，但很少有人探索核糖体失速如何在定量水平上影响翻译持续时间。我们采用了一种用于测量伸长时间的方法，并将其应用于酿酒酵母，以量化伸长停滞的影响。我们发现，在含有 Arg CGA 密码子重复诱导的停顿的转录本中，Hel2 介导的蛋白质表达和 mRNA 水平呈剂量依赖性下降，并且延伸延迟在几分钟左右。在包含非最佳 Leu 密码子同义取代的转录本中，蛋白质和 mRNA 水平下降，以及类似的延伸延迟，但这是通过非 Hel2 介导的机制发生的。最后，我们发现 Dhh1 选择性地增加蛋白质表达、mRNA 水平和延伸率。这表明，尽管延伸失速持续时间相似，但不同的翻译不良的 mRNA 将激活不同的救援途径。总而言之，这些结果为翻译的监控以及 Hel2 和 Dhh1 在介导核糖体暂停事件中的作用提供了新的定量机制见解。