Center for Molecular Biology of RNA, University of California, Santa Cruz, CA 95064.
Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, CA 95064.
Proc Natl Acad Sci U S A. 2022 Nov;119(44):e2212502119. doi: 10.1073/pnas.2212502119. Epub 2022 Oct 25.
Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3'-terminal pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.
转移 RNA(tRNA)和信使 RNA(mRNA)穿过核糖体的转位由细菌中的 GTP 酶延伸因子 G(EF-G)催化。尽管鸟苷-5'-三磷酸(GTP)水解加速了转位，并且 EF-G 的解离需要 GTP 水解，但它的基本作用仍然不清楚。在这里，我们使用整体Förster 共振能量转移(FRET)来监测 GTP 水解抑制如何影响核糖体的结构动力学。我们使用了 S12-S19 和 S11-S13 的 FRET 对，它们明确报告 30S 头部结构域的旋转，以及 S6-L9 对，它测量亚基间的旋转。我们的结果表明，除了以前显示的减缓反向亚基间旋转外，阻止 GTP 水解还会减缓正向头部旋转。令人惊讶的是，完全阻止 GTP 水解会完全消除反向头部旋转。我们发现，以前用于监测头部旋转的 S13-L33 FRET 对似乎几乎专门报告亚基间旋转。此外，我们发现，广泛用于跟踪 mRNA 转位的 3'-末端芘标记 mRNA 猝灭的信号与反向亚基间旋转最相关。为了解释我们的发现，即阻止 GTP 水解会消除在 mRNA 和 tRNA 的运动基本完成后发生的旋转事件，我们提出 GTP 水解的主要作用是在一种机制中创建不可逆步骤，该机制防止 EF-G 释放，直到 tRNA 和 mRNA 都移动了一个完整的密码子，从而确保有效的转位和翻译阅读框的维持。
