Directional transit of the ribosome along the messenger RNA (mRNA) template is a key determinant of the rate and processivity of protein synthesis. Imaging of the multistep translocation mechanism using single-molecule FRET has led to the hypothesis that substrate movements relative to the ribosome resolve through relatively long-lived late intermediates wherein peptidyl-tRNA enters the P site of the small ribosomal subunit via reversible, swivel-like motions of the small subunit head domain within the elongation factor G (GDP)-bound ribosome complex. Consistent with translocation being rate-limited by recognition and productive engagement of peptidyl-tRNA within the P site, we now show that base-pairing mismatches between the peptidyl-tRNA anticodon and the mRNA codon dramatically delay this rate-limiting, intramolecular process. This unexpected relationship between aminoacyl-tRNA decoding and translocation suggests that miscoding antibiotics may impact protein synthesis by impairing the recognition of peptidyl-tRNA in the small subunit P site during EF-G–catalyzed translocation. Strikingly, we show that elongation factor P (EF-P), traditionally known to alleviate ribosome stalling at polyproline motifs, can efficiently rescue translocation defects arising from miscoding. These findings help reveal the nature and origin of the rate-limiting steps in substrate translocation on the bacterial ribosome and indicate that EF-P can aid in resuming translation elongation stalled by miscoding errors.

中文翻译：

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错误编码导致细菌核糖体上底物易位的失速

核糖体沿信使RNA（mRNA）模板的方向转移是决定蛋白质合成速率和合成能力的关键因素。使用单分子FRET进行多步易位机制的成像已得出这样的假说，即相对于核糖体的底物运动通过相对长寿的晚期中间体而分解，其中肽基-tRNA通过可逆的，旋转样进入小核糖体亚基的P位点伸长因子G（GDP）结合的核糖体复合物中小亚基头部域的运动。与通过在P位点内对肽基-tRNA的识别和生产性参与来限制速率的一致，我们现在表明，肽基-tRNA反密码子和mRNA密码子之间的碱基配对错配极大地延迟了该限速分子内过程。氨酰基-tRNA解码与易位之间的这种出乎意料的关系表明，错误编码的抗生素可能会削弱在EF-G催化的易位过程中小亚基P位点对肽基-tRNA的识别，从而影响蛋白质的合成。令人惊讶的是，我们显示了传统上已知的可减轻多脯氨酸基序上的核糖体停滞的延伸因子P（EF-P），可以有效地挽救因编码错误而引起的易位缺陷。这些发现有助于揭示细菌核糖体上底物易位的限速步骤的性质和起源，并表明EF-P可以帮助恢复因错误编码错误而停滞的翻译延伸。