Probing the function of translational pausing in bacterial protein synthesis

探讨细菌蛋白质合成中翻译暂停的功能

• 批准号: 8653973
• 负责人: Gene-Wei Li
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2015-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8653973
• 关键词: Address; Automobile Driving; Bacteria; Bacterial Proteins; Biochemical; Biochemistry; Biological Assay; Cells; Cellular biology; Codon Nucleotides; Complement; Computing Methodologies; Controlled Study; Cyanobacterium; Data; Defect; Engineering; Escherichia coli; Eukaryota; Exhibits; Gene Expression; Gene Proteins; Genes; Goals; Growth; Investigation; Kinetics; Knowledge; Life; Measurement; Membrane; Mentors; Messenger RNA; Molecular Motors; Noise; Operon; Optics; Organism; Pattern; Pharmacologic Substance; Phase; Physics; Play; Post-Transcriptional Regulation; Process; Prokaryotic Cells; Protein Biosynthesis; Proteins; Repression; Research; Resolution; Resources; Ribosomal RNA; Ribosomes; Role; Site; Solid; Speed; Stress; Synechococcus; System; Technology; Testing; Thinking; Time; Training; Translation Initiation; Translational Repression; Translations; Transmembrane Domain; Variant; Work; biological adaptation to stress; cellular imaging; deep sequencing; drug production; genome sequencing; genome-wide; in vivo; knowledge base; novel; programs; protein expression; protein folding; public health relevance; skills

DESCRIPTION (provided by applicant): The speed of protein synthesis can impact all co-translational processes, from folding to degradation of the nascent chain. It was not until 3 years ago that we had the first global views of the speed of translation with sub-codon resolution in vivo. The enabling technology is ribosome profiling-deep sequencing of ribosome-protected mRNA fragments-developed in the Weissman lab at UCSF. By combining ribosome profiling with computational approaches, I have now initiated an effort to decipher how translational pausing regulates protein synthesis. Since starting at UCSF, I made the surprising discovery that the majority of translational pause sites in bacteria occur at internal Shine-Dalgarno (SD) sequences, driven by their interaction with the anti-Shine-Dalgarno (antiSD) region of the elongating ribosome. The current paradigm, established by Shine and Dalgarno in 1975, is that the main role of the ribosomal antiSD region is to define translation initiation sites in prokaryotes. My finding that there is conserved and ubiquitous pausing at internal SD sites suggests a distinct function for the antiSD region during the elongation phase of translation. In fact, recent genome sequencing data have revealed that, although the antiSD region of ribosomal RNA is highly conserved throughout prokaryotes, many bacterial and archaeal species do not use it for translation initiation. Intriguingly, several intragenic SD sites are conserved across many species. I hypothesize that this novel function of antiSD during translational elongation is an important factor driving the conservation of the antiSD region. Objective: To understand the widespread use of SD-induced pausing, I propose to investigate the co- translational processes that are controlled by pausing sites identified by our genome-wide measurements. My immediate goals are to define the broader role of anti-Shine-Dalgarno sequence in prokaryotic translation, and to determine the role of translational pausing in protein folding, membrane insertion, and post-transcriptional regulation. This work will elucidate the principles governing the interplay between translational pausing and co- translational processes in all organisms including eukaryotes, which also exhibit ubiquitous, albeit mechanistically distinct pauses with unexplored functions. Coming from a background in physics, I am seeking to complement my analytical and optical skills with solid hands-on training in cell biology and biochemistry. In pursuit of these aims with my mentors' expertise in protein folding and stress responses, I will acquire both the knowledge base and a unique perspective to launch my own independent investigation on gene expression and protein synthesis from the mechanistic level to the systems level.

描述(由申请人提供)：蛋白质合成的速度可以影响所有的共翻译过程，从折叠到新生链条的降解。直到三年后 在此之前，我们第一次对体内亚密码子分辨率的翻译速度进行了全球观察。使之成为可能的技术是由加州大学旧金山分校的魏斯曼实验室开发的核糖体图谱分析--对核糖体保护的mRNA片段进行深度测序。通过将核糖体分析与计算方法相结合，我现在已经开始了一项努力，以破译翻译停顿如何调节蛋白质合成。自从开始在加州大学旧金山分校工作以来，我有了一个令人惊讶的发现，细菌中的大多数翻译停顿位点都发生在内部的Shine-Dalgarno(SD)序列中，这是由它们与细长核糖体的反Shine-Dalgarno(AntiSD)区域相互作用所驱动的。目前的范式是由Shine和Dalgarno于1975年建立的，即核糖体抗SD区域的主要作用是定义原核生物中的翻译起始点。我的发现表明，在内部SD位点存在保守且普遍存在的停顿，这表明在翻译的延伸阶段，抗SD区域具有独特的功能。事实上，最近的基因组测序数据表明，尽管核糖体RNA的抗SD区域在原核生物中高度保守，但许多细菌和古生物物种并不利用它来启动翻译。有趣的是，几个基因内SD位点在许多物种中都是保守的。我推测，抗SD在翻译延伸过程中的这种新功能是推动抗SD区域保守的一个重要因素。目的：为了了解SD诱导的停顿的广泛使用，我建议研究由我们的全基因组测量确定的停顿位点控制的共翻译过程。我的近期目标是确定反Shine-Dalgarno序列在原核细胞翻译中的广泛作用，并确定翻译暂停在蛋白质折叠、膜插入和转录后调节中的作用。这项工作将阐明在包括真核生物在内的所有生物体中，翻译暂停和共翻译过程之间相互作用的原理，真核生物也表现出无处不在的，尽管机械上截然不同的暂停，但功能尚未被探索。我有物理学的背景，希望通过在细胞生物学和生物化学方面的扎实实践培训来补充我的分析和光学技能。为了实现这些目标，凭借我的导师在蛋白质折叠和应激反应方面的专业知识，我将获得知识基础和独特的视角，从机制层面到系统层面展开自己对基因表达和蛋白质合成的独立研究。