Structural Basis for the Allosteric Mechanisms Regulating Ribosome Function

调节核糖体功能的变构机制的结构基础

• 批准号: 10266166
• 负责人: MATTHIEU GAGNON
• 金额: $ 33.18万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-18 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10266166
• 关键词: Adopted; Allosteric Site; Amino Acids; Amino Acyl Transfer RNA; Antibiotics; Anticodon; Bacteria; Base Pair Mismatch; Base Pairing; Binding; Biochemical; Biological Assay; Clinical; Codon Nucleotides; Complex; Cryoelectron Microscopy; Data; Development; Dissociation; Elements; Ensure; Event; Foundations; Goals; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hydrolysis; Impairment; Infection; Initiator Codon; Initiator tRNA; Kinetics; Knowledge; Mediating; Messenger RNA; Methionine; Methods; Molecular; Molecular Conformation; Molecular Machines; Movement; Organism; Peptide Elongation Factor G; Peptides; Positioning Attribute; Process; Prokaryotic Initiation Factor-2; Property; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Pseudomonas aeruginosa; Quality Control; Reading Frames; Recycling; Reporting; Ribosomes; Role; Sense Codon; Signal Transduction; Site; Structure; Techniques; Terminator Codon; Testing; Thermus thermophilus; Transfer RNA; Translation Initiation; Translations; X-Ray Crystallography; base; design; experimental study; fighting; functional outcomes; human pathogen; improved; inhibitor/antagonist; insight; interdisciplinary approach; molecular clock; next generation; novel; novel therapeutics; pathogenic bacteria; peptidyl-tRNA; polypeptide; release factor; ribosome releasing factor; translation factor

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.

概括 核糖体是一种复杂的分子机，负责解码mRNA并产生所有蛋白质 在每个生物体中。该过程需要选择trnas，胡椒键的形成，tRNA运动。 密码子每个伸长循环以及多肽链的释放。翻译因素是关键调节因素 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 功能从阐明翻译机制的结构方法受益匪浅 分子水平终止期间的伸长和停止密码子识别。因为核糖体是 大多数临床上有用的抗生素的靶标，核糖体的许多结构与因素和因素和 抑制剂允许开发出色的抗生素。但是，值得注意的是，两个机制 蛋白质合成，启动和核糖体回收的最重要步骤尚不清楚。 核糖体转化和回收为亚基标记了蛋白质的开头和末端 合成周期，因此可以更好地理解这些过程的分子方面 新疗法的门。我们最近的发现揭示了翻译启动之间的相似之处 和核糖体回收：在这两个步骤中，肽基（P）部位中的tRNA采用了高度相似的构象 由翻译因子诱导。尽管如此，密码子 - 古典互动的命运必须不同 因为在翻译启动过程中，启动密码子由启动器tRNA识别，在回收过程中， P网站中的密码子 - 抗病基础配对预计将受到破坏。这表明基本状态 mRNA和P位点之间的配对是核糖体功能的主要控制元素，这是一个方面 到目前为止被忽视的翻译。为了了解核糖体的分子机制 回收和翻译计划，我们建议研究翻译的非常规方面。因此，在AIM 1中， 我们将确定人病原体假单胞菌中核糖体回收的分子机制 由非正统伸长因子G-1A（EF-G1A）制备的铜绿菌，这是一种专门的EF-G 仅在核糖体回收中起作用。在AIM 2中，我们将确定P.中的主动性因子2（IF2）如何。 铜绿菌与国有居住的配方状态独立于识别引发剂tRNA。目标 3，我们将表征P站点中使用启动器TRNA的密码子 - 抗原mispair如何变构触发器 核糖体的“质量检查”改变了氨基酰基（a）位点的解码特性。这些目标会 可以使用多学科方法来完成，包括最先进的冷冻电子显微镜（冷冻 - EM）和X射线晶体学的大型核糖体配合物，以及生化方法 由于停止流动动力学实验和核糖体结合测定。预期的发现将填补重要空白 了解核糖体功能，并可能为结构引导的发展提供未经证实的机会 蛋白质合成的新抑制剂。