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

基本信息

批准号：
9207011
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
项目类别：
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/9207011
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 Genetic Transcription Goals Growth Investigation Kinetics Knowledge 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 Protein Engineering 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 ribosome profiling skills

项目摘要

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/摘要理由：蛋白质合成的速度可能会影响所有共同过程，从折叠到新生链的降解。直到3年前，我们才对速度的第一个全球观点带有亚果酱分辨率在体内的翻译。促成技术是核糖体分析 - 深度测序由核糖体保护的mRNA片段 - 在UCSF的魏斯曼实验室中开发。通过结合核糖体通过计算方法进行分析，我现在开始努力破译翻译的暂停调节蛋白质合成。自从UCSF开始以来，我惊讶地发现，大多数翻译停顿网站细菌发生在内部光泽 - 达尔加诺（SD）序列中，由它们与抗新的相互作用驱动伸长核糖体的Dalgarno（AntISD）区域。当前的范式，由Shine和达尔诺（Dalgarno）于1975年，核糖体抗体区域的主要作用是定义翻译起始位点原核生物。我发现在内部SD站点有保守和无处不在的暂停表明一个明显的在翻译的伸长阶段，AntISD区域的功能。实际上，最近的基因组测序数据表明，尽管核糖体RNA的抗抗Antisd区域高度构成原核生物，许多细菌和古细菌物种都不将其用于翻译启动。有趣的是，有几个基因内SD位点在许多物种中都保存。我假设在翻译伸长是推动AntISD区域保护的重要因素。目的：了解SD引起的暂停的广泛使用，我建议研究共同通过我们全基因组测量确定的暂停位点控制的翻译过程。我的直接的目标是定义抗新dalgarno序列在原核翻译中的更广泛作用，并且确定平移暂停在蛋白质折叠，膜插入和翻译后的作用规定。这项工作将阐明管理翻译暂停与共同相互作用的原则包括真核生物在内的所有生物的翻译过程，它们也无处不在，尽管机械上不同的暂停，具有意外的功能。我来自物理背景，我正在寻求通过细胞生物学和生物化学方面的实践训练。为了追求这些目标，我的导师的专业知识蛋白质折叠和压力反应，我将获得知识库和独特的视角从机械水平开始我自己对基因表达和蛋白质合成的独立投资到系统级别。