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: 8690897
• 负责人: Christopher S. Hayes
• 金额: $ 26.51万
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8690897
• 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)： 这项研究项目的长期目标是了解细胞如何在蛋白质合成过程中识别和回收非活性核糖体。并不是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，蛋白质合成的不完全是由于“不停止”的mRNAs，它是缺少框架内终止密码子的截短转录本。在细菌中，困在不间断信使核糖核酸34端的核糖体被转移信使核糖核酸质量控制系统“拯救”。虽然tmRNA在整个真细菌中普遍存在，但由于存在一条平行的tmRNA非依赖核糖体拯救途径，它可以从大肠杆菌细胞中删除。然而，这两条途径在大肠杆菌中都不能被破坏，这表明核糖体拯救对这些细胞乃至所有细胞都是必不可少的功能。这项建议侧重于围绕核糖体拯救的翻译质量控制的三个基本方面。首先，我们试图确定A-位点mRNA切割和核糖体拯救之间的功能关系。A位裂解是一种新的核糖核酸酶活性，它能截断停滞核糖体A位密码子中的mRNA，从而产生不间断的mRNA。将使用生化和分子遗传学方法来鉴定A位核酸酶。鉴定A-位核糖核酸酶对于确定A-位核糖核酸酶的活性在tmRNA介导的核糖体营救或非tmRNA非依赖核糖体营救中起作用是必要的。其次，我们将描述最近发现的tmRNA非依赖的救援途径，它似乎是由YhdL介导的。YhdL的生化功能和调节将通过体外和体内方法进行研究。将确定YhdL核糖体拯救活性的谱，并确定其在新生链释放中的作用。最后，将从机制上确定DNAK在tmRNA介导的核糖体拯救中的作用。DNAK活性将通过分子遗传学和药理学方法被去除，并对tmRNA标记的影响进行评估。一个明确的体外翻译系统将被用来确定DNAK是否通过其共翻译伴侣活性发挥其作用。

项目成果

Deletion of the RluD pseudouridine synthase promotes SsrA peptide tagging of ribosomal protein S7.

• DOI: 10.1111/j.1365-2958.2010.07467.x
• 发表时间: 2011-01
• 期刊: Molecular microbiology
• 影响因子: 3.6
• 作者: Schaub RE;Hayes CS
• 通讯作者: Hayes CS

Analysis of aminoacyl- and peptidyl-tRNAs by gel electrophoresis.

• DOI: 10.1007/978-1-61779-949-5_19
• 发表时间: 2012
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Janssen, Brian D;Diner, Elie J;Hayes, Christopher S
• 通讯作者: Hayes, Christopher S

Receptor polymorphism restricts contact-dependent growth inhibition to members of the same species.

• DOI: 10.1128/mbio.00480-13
• 发表时间: 2013-07-23
• 期刊: mBio
• 影响因子: 6.4
• 作者: Ruhe ZC;Wallace AB;Low DA;Hayes CS
• 通讯作者: Hayes CS

YoeB toxin is activated during thermal stress.

• DOI: 10.1002/mbo3.272
• 发表时间: 2015-08
• 期刊: MicrobiologyOpen
• 影响因子: 3.4
• 作者: Janssen BD;Garza-Sánchez F;Hayes CS
• 通讯作者: Hayes CS

The N-terminus of GalE induces tmRNA activity in Escherichia coli.

• DOI: 10.1371/journal.pone.0015207
• 发表时间: 2010-12-07
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Ruhe ZC;Hayes CS
• 通讯作者: Hayes CS