Structural Basis for the Allosteric Mechanisms Regulating Ribosome Function

总结 核糖体是一个复杂的分子机器，负责解码mRNA并产生所有蛋白质 在每一个有机体。这一过程需要选择tRNA，形成肽键，tRNA的运动， 密码子每个延长周期，并释放多肽链。翻译因子是 核糖体功能，调节核糖体本身和tRNA的构象。我们对核糖体的了解 功能学从阐明翻译机制的结构方法中获益匪浅 在分子水平终止期间的延伸和终止密码子识别。因为核糖体是 作为大多数临床上有用的抗生素的靶点，核糖体的许多结构与因子复合， 抑制剂使得上级抗生素的开发成为可能。然而，值得注意的是， 蛋白质合成、起始和核糖体再循环的最重要步骤仍然不清楚。 翻译起始和核糖体再循环成亚基标志着蛋白质的开始和结束 合成周期，因此，更好地了解这些过程的分子方面可以打开 新疗法的大门我们最近的发现揭示了翻译起始和翻译起始之间 和核糖体再循环：在这两个步骤中，肽基（P）位点的tRNA采用高度相似的构象， 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一定是不同的 因为在翻译起始期间，起始密码子被起始tRNA识别，并且在再循环期间， 预期P位点中的密码子-反密码子碱基配对被破坏。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， 这是迄今为止一直被忽视的翻译。深入了解核糖体的分子机制 回收和翻译启动，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中核糖体再循环的分子机制 非正统延伸因子G-1A（EF-G1 A）是一种专门的EF-G， 只在核糖体循环中起作用。在目标2中，我们将确定起始因子2（IF 2）如何在P。 铜绿假单胞菌识别起始tRNA，而不依赖于甲硫氨酸残基的构象。在Aim中 3，我们将描述密码子-反密码子错配与起始tRNA在P位点的变构触发 核糖体的“质量检查”改变了氨酰基（A）位点的解码特性。这些目标将 使用多学科方法完成，包括最先进的冷冻电子显微镜（cryo-electron microscopy， EM）和X射线晶体学，以及生物化学方法， 作为停流动力学实验和核糖体结合测定。预期的调查结果将填补重要的空白 在核糖体功能的知识，并可能提供意想不到的机会，结构指导的发展 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.