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Precise tuning of bacterial translation initiation by non-equilibrium 5′-UTR unfolding observed in single mRNAs
Nucleic Acids Research ( IF 14.9 ) Pub Date : 2022-07-27 , DOI: 10.1093/nar/gkac635
Sujay Ray 1 , Shiba S Dandpat 1 , Surajit Chatterjee 1 , Nils G Walter 1
Affiliation  

Noncoding, structured 5′-untranslated regions (5′-UTRs) of bacterial messenger RNAs (mRNAs) can control translation efficiency by forming structures that either recruit or repel the ribosome. Here we exploit a 5′-UTR embedded preQ1-sensing, pseudoknotted translational riboswitch to probe how binding of a small ligand controls recruitment of the bacterial ribosome to the partially overlapping Shine-Dalgarno (SD) sequence. Combining single-molecule fluorescence microscopy with mutational analyses, we find that the stability of 30S ribosomal subunit binding is inversely correlated with the free energy needed to unfold the 5′-UTR during mRNA accommodation into the mRNA binding cleft. Ligand binding to the riboswitch stabilizes the structure to both antagonize 30S recruitment and accelerate 30S dissociation. Proximity of the 5′-UTR and stability of the SD:anti-SD interaction both play important roles in modulating the initial 30S-mRNA interaction. Finally, depletion of small ribosomal subunit protein S1, known to help resolve structured 5′-UTRs, further increases the energetic penalty for mRNA accommodation. The resulting model of rapid standby site exploration followed by gated non-equilibrium unfolding of the 5′-UTR during accommodation provides a mechanistic understanding of how translation efficiency is governed by riboswitches and other dynamic structure motifs embedded upstream of the translation initiation site of bacterial mRNAs.

中文翻译:

通过在单个 mRNA 中观察到的非平衡 5'-UTR 展开精确调节细菌翻译起始

细菌信使 RNA (mRNA) 的非编码、结构化 5'-非翻译区 (5'-UTR) 可以通过形成招募或排斥核糖体的结构来控制翻译效率。在这里,我们利用 5'-UTR 嵌入 preQ1 感应、假结翻译核糖开关来探测小配体的结合如何控制细菌核糖体向部分重叠的 Shine-Dalgarno (SD) 序列的募集。将单分子荧光显微镜与突变分析相结合,我们发现 30S 核糖体亚基结合的稳定性与在 mRNA 适应过程中将 5'-UTR 展开到 mRNA 结合间隙中所需的自由能呈负相关。与核糖开关结合的配体稳定了结构,既能拮抗 30S 募集,又能加速 30S 解离。5'-UTR 的接近性和 SD:anti-SD 相互作用的稳定性都在调节初始 30S-mRNA 相互作用中起重要作用。最后,小核糖体亚基蛋白 S1 的消耗,已知有助于解决结构化的 5'-UTR,进一步增加了 mRNA 调节的能量损失。由此产生的快速备用位点探索模型,随后在调节期间对 5'-UTR 进行门控非平衡展开,提供了对翻译效率如何由嵌入细菌 mRNA 翻译起始位点上游的核糖开关和其他动态结构基序控制的机械理解. 进一步增加了 mRNA 调节的能量损失。由此产生的快速备用位点探索模型,随后在调节期间对 5'-UTR 进行门控非平衡展开,提供了对翻译效率如何由嵌入细菌 mRNA 翻译起始位点上游的核糖开关和其他动态结构基序控制的机械理解. 进一步增加了 mRNA 调节的能量损失。由此产生的快速备用位点探索模型,随后在调节期间对 5'-UTR 进行门控非平衡展开,提供了对翻译效率如何由嵌入细菌 mRNA 翻译起始位点上游的核糖开关和其他动态结构基序控制的机械理解.
更新日期:2022-07-27
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