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: 9002063
• 负责人: Gene-Wei Li
• 金额: $ 24.9万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 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 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9002063
• 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; abstracting; biological adaptation to stress; cellular imaging; deep sequencing; drug production; genome sequencing; genome-wide; in vivo; knowledge base; novel; programs; protein expression; protein folding; ribosome profiling; skills

Project Summary/Abstract Rationale: 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.

项目摘要/摘要 原理：蛋白质合成的速度可以影响所有的共翻译过程，从折叠到 新生的链条的退化。直到3年前，我们才第一次在全球范围内了解 体内亚密码子解析翻译。使之成为可能的技术是核糖体图谱--深度测序 核糖体保护的信使核糖核酸片段-在加州大学旧金山分校的魏斯曼实验室开发。通过结合核糖体 使用计算方法进行分析，我现在已经开始努力破译翻译停顿是如何 调节蛋白质的合成。 自从开始在加州大学旧金山分校工作以来，我有一个令人惊讶的发现，大多数翻译停顿站点在 细菌出现在Shine-Dalgarno(SD)内部序列中，受它们与反Shine-Dalgarno(SD)序列相互作用的驱动。 伸长核糖体的Dalgarno(抗SD)区域。目前的范式，由Shine和 Dalgarno在1975年提出的，核糖体抗SD区域的主要作用是定义翻译起始点 原核生物。我的发现是，在内部SD站点存在保守且普遍的停顿，这表明了一种独特的 在翻译的延伸阶段，抗SD区域的功能。事实上，最近的基因组测序 数据显示，尽管核糖体RNA的抗SD区域在整个过程中都高度保守 原核生物，许多细菌和古生物物种不使用它来启动翻译。耐人寻味的是，有几个 基因内SD位点在许多物种中都是保守的。我推测抗SD的这一新功能在 翻译延长是驱动抗SD区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，我建议调查 由我们的全基因组测量确定的停顿位点控制的翻译过程。我的 近期的目标是确定反Shine-Dalgarno序列在原核翻译中的更广泛作用，以及 确定翻译暂停在蛋白质折叠、膜插入和转录后的作用 监管。这项工作将阐明翻译暂停和协同翻译之间相互作用的原则。 所有生物体中的翻译过程，包括真核生物，也表现出无处不在的，尽管 机械上截然不同的停顿和未探索的功能。 我有物理学的背景，我想通过以下方式来补充我的分析和光学技能 扎实的细胞生物学和生物化学方面的实践培训。为了实现这些目标，我的导师们在 蛋白质折叠和应激反应，我将获得这两个知识库和一个独特的视角 从机制层面对基因表达和蛋白质合成展开自己的独立研究 到系统层面。