Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
Department of Chemistry, University of Michigan, Ann Arbor, Michigan, USA.
J Biol Chem. 2022 Jun;298(6):102039. doi: 10.1016/j.jbc.2022.102039. Epub 2022 May 17.
Ribosome speed is dictated by multiple factors including substrate availability, cellular conditions, and product (peptide) formation. Translation slows during the synthesis of cationic peptide sequences, potentially influencing the expression of thousands of proteins. Available evidence suggests that ionic interactions between positively charged nascent peptides and the negatively charged ribosome exit tunnel impede translation. However, this hypothesis was difficult to test directly because of inability to decouple the contributions of amino acid charge from mRNA sequence and tRNA identity/abundance in cells. Furthermore, it is unclear if other components of the translation system central to ribosome function (e.g., RNA modification) influence the speed and accuracy of positively charged peptide synthesis. In this study, we used a fully reconstituted Escherichia coli translation system to evaluate the effects of peptide charge, mRNA sequence, and RNA modification status on the translation of lysine-rich peptides. Comparison of translation reactions on poly(lysine)-encoding mRNAs conducted with either Lys-tRNA or Val-tRNA reveals that that amino acid charge, while important, only partially accounts for slowed translation on these transcripts. We further find that in addition to peptide charge, mRNA sequence and both tRNA and mRNA modification status influence the rates of amino acid addition and the ribosome's ability to maintain frame (instead of entering the -2, -1, and +1 frames) during poly(lysine) peptide synthesis. Our observations lead us to expand the model for explaining how the ribosome slows during poly(lysine) peptide synthesis and suggest that posttranscriptional RNA modifications can provide cells a mechanism to precisely control ribosome movements along an mRNA.
核糖体的速度受到多种因素的制约，包括底物的可利用性、细胞状态和产物（肽）的形成。在阳离子肽序列合成过程中，翻译会减慢，这可能会影响数千种蛋白质的表达。现有证据表明，带正电荷的新生肽与带负电荷的核糖体出口通道之间的离子相互作用会阻碍翻译。然而，由于无法将氨基酸电荷与 mRNA 序列和 tRNA 的身份/丰度在细胞中分离出来，这个假设很难直接进行测试。此外，尚不清楚翻译系统的其他组成部分（如 RNA 修饰）是否会影响带正电荷的肽合成的速度和准确性。在这项研究中，我们使用完全重组的大肠杆菌翻译系统来评估肽电荷、mRNA 序列和 RNA 修饰状态对富含赖氨酸的肽翻译的影响。用 Lys-tRNA 或 Val-tRNA 对编码多聚赖氨酸的 mRNA 进行翻译反应的比较表明，虽然氨基酸电荷很重要，但它仅部分解释了这些转录物翻译速度减慢的原因。我们进一步发现，除了肽电荷外，mRNA 序列以及 tRNA 和 mRNA 的修饰状态都会影响氨基酸的添加速度和核糖体在多聚赖氨酸肽合成过程中保持框架（而不是进入-2、-1 和+1 框架）的能力。我们的观察结果使我们扩大了解释核糖体在多聚赖氨酸肽合成过程中减速的模型，并表明转录后 RNA 修饰可以为细胞提供一种精确控制核糖体沿 mRNA 运动的机制。
