Structural Basis for the Allosteric Mechanisms Regulating Ribosome Function

摘要 核糖体是一个复杂的分子机器，负责解码信使核糖核酸并产生所有蛋白质。 在每一种有机体中。这个过程需要tRNA的选择，多肽键的形成，tRNA的移动 每个延伸循环密码子，并释放多肽链。翻译因素是关键的调节因素 核糖体的功能，调节核糖体本身和tRNA的构象。我们对核糖体的了解 功能从阐述翻译机制的结构方法中受益匪浅 终止过程中在分子水平上的延伸和终止密码子识别。因为核糖体是 作为大多数临床使用抗生素的靶标，核糖体的许多结构与这些因子和 抑制剂使优质抗生素的开发成为可能。然而，值得注意的是，其中两种机制 蛋白质合成的最重要步骤，即启动和核糖体循环，仍然不清楚。 核糖体的翻译起始和亚基的再循环标志着蛋白质的开始和结束 合成循环，因此更好地理解这些过程的分子方面可以打开 通往新疗法的大门。我们最近的发现揭示了翻译启动与翻译之间的相似之处 和核糖体循环：在这两个步骤中，肽(P)位点的tRNA采用高度相似的构象，即 是由翻译因素引起的。尽管如此，密码子-反密码子相互作用的命运肯定是不同的 因为在翻译启动过程中，起始密码子被启动子tRNA识别，而在循环过程中， 预计P位点的密码子-反密码子碱基配对可能会中断。这表明基地的状态 MRNA和P-位点tRNA之间的配对是核糖体功能的主要控制元件，这是 到目前为止一直被忽视的翻译。深入了解核糖体的分子机制 再循环和翻译启动，我们建议研究翻译的非常规方面。因此，在目标1中， 我们将确定人类病原体假单胞菌核糖体循环的分子机制 由非常规延长因子G-1a(EF-G1a)促进的铜绿假单胞菌，一种特殊的EF-G 在核糖体循环中独一无二地发挥作用。在目标2中，我们将确定启动因子2(IF2)在P. 铜绿假单胞菌独立于甲硫氨酸残基的甲酰化状态识别启动子tRNA。在AIM 3，我们将描述密码子-反密码子错配如何与P位点上的启动子tRNA变构触发 由核糖体进行的“质量检查”，它改变了氨基酰(A)位点的解码特性。这些目标将 使用多学科方法完成，包括最先进的低温电子显微镜(低温电子显微镜) EM)和X射线结晶学，以及生物化学方法，如 作为停流动力学实验和核糖体结合实验。预期的发现将填补重要的空白。 在核糖体功能方面的知识，并可能为结构引导的发育提供意想不到的机会 新的蛋白质合成抑制剂。

SUMMARY The ribosome is a complex molecular machine responsible for decoding the mRNA and producing all proteins in every organism. The process entails the selection of tRNAs, peptide bond formation, tRNA movement by one codon each elongation cycle, and release of the polypeptide chain. Translation factors are key regulators of ribosome function, modulating the conformation of the ribosome itself and of tRNAs. Our knowledge of ribosome functioning has benefited immensely from structural approaches that elucidated mechanisms of translation elongation and stop codon recognition during termination at a molecular level. Because the ribosome is the target for most of the clinically useful antibiotics, many structures of the ribosome in complex with the factors and inhibitors have allowed development of superior antibiotics. Remarkably, however, the mechanisms for two of the most important steps of protein synthesis, initiation and ribosome recycling, have remained unclear. Translation initiation and recycling of the ribosome into subunits mark the beginning and the end of the protein synthesis cycle, and therefore a better understanding of the molecular aspects of these processes could open the door to new therapeutics. Our recent findings reveal an unsuspected similarity between translation initiation and ribosome recycling: in both steps, the tRNA in the peptidyl (P) site adopts a highly similar conformation that is induced by translation factors. Despite this, the fate of the codon-anticodon interaction must be different because during translation initiation, the start codon is recognized by the initiator tRNA and during recycling, the codon-anticodon base pairing in the P site is expected to be disrupted. This suggests that the state of base pairing between the mRNA and the P-site tRNA is a major control element of ribosome functioning, an aspect of translation that has been so far overlooked. To gain insights into the molecular mechanisms of ribosome recycling and translation initiation, we propose to study unconventional aspects of translation. Hence, in Aim 1, we will determine the molecular mechanism of ribosome recycling in the human pathogen Pseudomonas aeruginosa that is facilitated by the unorthodox elongation factor G-1A (EF-G1A), a specialized EF-G that exclusively functions in ribosome recycling. In Aim 2, we will determine how initiation factor 2 (IF2) in P. aeruginosa recognizes the initiator tRNA independently of the formylation state of the methionine residue. In Aim 3, we will characterize how a codon-anticodon mispair with the initiator tRNA in the P site allosterically triggers a “quality check” by the ribosome that alters the decoding properties of the aminoacyl (A) site. These aims will be accomplished using multidisciplinary approaches, including state-of-the-art cryo-electron microscopy (cryo- EM) and X-ray crystallography of large functional ribosome complexes, together with biochemical methods such as stopped flow kinetic experiments and ribosome binding assays. The anticipated findings will fill important gaps in knowledge of ribosome functioning and may offer unsuspected opportunities for structure-guided development of new inhibitors of protein synthesis.