Inhibitory codon pairs and poly(A) tracts within the translated mRNA cause ribosome stalling and reduce protein output. The molecular mechanisms that drive these stalling events, however, are still unknown. Here, we use a combination of in vitro biochemistry, ribosome profiling, and cryo-EM to define molecular mechanisms that lead to these ribosome stalls. First, we use an in vitro reconstituted yeast translation system to demonstrate that inhibitory codon pairs slow elongation rates which are partially rescued by increased tRNA concentration or by an artificial tRNA not dependent on wobble base-pairing. Ribosome profiling data extend these observations by revealing that paused ribosomes with empty A sites are enriched on these sequences. Cryo-EM structures of stalled ribosomes provide a structural explanation for the observed effects by showing decoding-incompatible conformations of mRNA in the A sites of all studied stall- and collision-inducing sequences. Interestingly, in the case of poly(A) tracts, the inhibitory conformation of the mRNA in the A site involves a nucleotide stacking array. Together, these data demonstrate a novel mRNA-induced mechanisms of translational stalling in eukaryotic ribosomes.

中文翻译：

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抑制密码子组合和聚腺苷酸束导致翻译停滞的分子机制。


翻译后的 mRNA 内的抑制性密码子对和 Poly(A) 束会导致核糖体停滞并减少蛋白质输出。然而，驱动这些失速事件的分子机制仍然未知。在这里，我们结合使用体外生物化学、核糖体分析和冷冻电镜来定义导致这些核糖体停滞的分子机制。首先，我们使用体外重建的酵母翻译系统来证明抑制密码子对会减慢延伸率，这可以通过增加 tRNA 浓度或通过不依赖于摆动碱基配对的人工 tRNA 来部分挽救。核糖体分析数据通过揭示具有空 A 位点的暂停核糖体在这些序列上富集，扩展了这些观察结果。停滞核糖体的冷冻电镜结构通过显示所有研究的停滞和碰撞诱导序列的 A 位点中 mRNA 的解码不相容构象，为观察到的效应提供了结构解释。有趣的是，在 Poly(A) 束的情况下，A 位点 mRNA 的抑制构象涉及核苷酸堆积阵列。这些数据共同证明了真核核糖体中 mRNA 诱导的翻译停滞机制。