Molecular determinants of A-site mRNA cleavage during ribosome pausing

核糖体暂停期间 A 位 mRNA 裂解的分子决定因素

• 批准号: 8500345
• 负责人: Christopher S. Hayes
• 金额: $ 25.79万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8500345
• 关键词: Animal Model; Antibiotics; Bacteria; Biochemical; Biochemical Genetics; Cells; Cleaved cell; Codon Nucleotides; Escherichia coli; Eubacterium; Goals; In Vitro; Laboratories; Lead; Length; Mediating; Mediator of activation protein; Messenger RNA; Modeling; Molecular; Molecular Biology; Molecular Chaperones; Molecular Genetics; Pathway interactions; Peptide Hydrolases; Peptides; Pharmacotherapy; Play; Process; Protein Biosynthesis; Proteins; Quality Control; Recycling; Regulation; Research; Research Project Grants; Ribonucleases; Ribosomes; Role; Site; System; Terminator Codon; Tertiary Protein Structure; Testing; Transcript; Translations; antimicrobial drug; in vitro activity; in vivo; novel; nuclease; protein folding; response

DESCRIPTION (provided by applicant): The long-term objective of this research project is to understand how cells identify and recycle inactive ribosomes during protein synthesis. Not all ribosomes that initiate translation are able to complete synthesis of full-length proteins. In many instances, incomplete protein synthesis is due to "non-stop" mRNAs, which are truncated transcripts lacking in-frame stop codons. In bacteria, ribosomes trapped at the 34-ends of non-stop mRNA are "rescued" by the tmRNA (for transfer-messenger RNA) quality control system. Although tmRNA is ubiquitous throughout the eubacteria, it can be deleted from Escherichia coli cells due to the presence of a parallel tmRNA-independent ribosome rescue pathway. However, both pathways cannot be disrupted in E. coli, demonstrating that ribosome rescue is an essential function for these and probably all cells. This proposal focuses on three fundamental aspects of translational quality control that revolve around ribosome rescue. First, we seek to determine the functional relationship between A-site mRNA cleavage and ribosome rescue. A- site cleavage is a novel RNase activity that truncates mRNA in the A-site codon of stalled ribosomes, thereby producing non-stop mRNA. Biochemical and molecular genetic approaches will be used to identify the A-site nuclease. Identification of the A-site nuclease is necessary to determine whether the activity plays a functional role in either tmRNA-mediated or tmRNA-independent ribosome rescue. Second, we will characterize the recently identified tmRNA-independent rescue pathway, which appears to be mediated by YhdL. The biochemical function and regulation of YhdL will be investigated using in vitro and in vivo approaches. The spectrum of YhdL ribosome rescue activity will be identified, and its role in nascent chain release determined. Finally, the role of DnaK in tmRNA-mediated ribosome rescue will be mechanistically defined. DnaK activity will be ablated using molecular genetic and pharmacological approaches and the effects on tmRNA tagging assessed. A defined in vitro translation system will be used to determine whether DnaK exerts its effects by virtue of its co-translational chaperone activity.

描述（由申请人提供）： 该研究项目的长期目标是了解细胞在蛋白质合成过程中如何识别和回收无活性的核糖体。并非所有启动翻译的核糖体都能完成全长蛋白质的合成。在许多情况下，不完整的蛋白质合成是由于“不间断”mRNA 造成的，这些 mRNA 是缺乏框内终止密码子的截短转录本。在细菌中，被困在不间断 mRNA 34 端的核糖体会被 tmRNA（转移信使 RNA）质量控制系统“拯救”。尽管 tmRNA 在真细菌中普遍存在，但由于存在平行的 tmRNA 独立核糖体救援途径，它可以从大肠杆菌细胞中删除。然而，这两种途径在大肠杆菌中都不能被破坏，这表明核糖体救援对于这些细胞甚至可能是所有细胞来说是一个重要功能。该提案重点关注围绕核糖体救援的翻译质量控制的三个基本方面。首先，我们试图确定 A 位 mRNA 切割和核糖体拯救之间的功能关系。 A 位点切割是一种新型 RNase 活性，可截断停滞核糖体 A 位密码子中的 mRNA，从而产生不间断的 mRNA。生化和分子遗传学方法将用于鉴定 A 位核酸酶。 A 位点核酸酶的鉴定对于确定该活性是否在 tmRNA 介导的或不依赖 tmRNA 的核糖体拯救中发挥功能作用是必要的。其次，我们将描述最近发现的不依赖于 tmRNA 的救援途径，该途径似乎是由 YhdL 介导的。将使用体外和体内方法研究 YhdL 的生化功能和调节。 YhdL 核糖体救援活性的范围将被确定，并确定其在新生链释放中的作用。最后，DnaK 在 tmRNA 介导的核糖体拯救中的作用将从机制上得到定义。将使用分子遗传学和药理学方法消除 DnaK 活性，并评估对 tmRNA 标记的影响。将使用确定的体外翻译系统来确定 DnaK 是否通过其共翻译伴侣活性发挥其作用。