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：
8487992
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8487992
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

项目摘要

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能会影响从新生链的折叠到降解的所有共同过程。直到3年才以前，我们对体内的亚果酱分辨率对翻译速度有了第一个全球观点。促成技术是在UCSF的魏斯曼实验室开发的核糖体保护的mRNA片段的核糖体分析深度测序。通过将核糖体分析与计算方法相结合，我现在开始努力破译转化如何调节蛋白质合成。自从在UCSF开始以来，我发现了一个令人惊讶的发现，细菌中的大多数翻译暂停位点发生在内部Shine-dalgarno（SD）序列，这是由于它们与伸出核糖体的抗新dalgarno（Antisd）区域的相互作用所驱动。当前由Shine和Dalgarno于1975年建立的范式是，核糖体抗Antisd区域的主要作用是定义原核生物中的翻译起始位点。我的发现，在内部SD位置存在保守和普遍存在的暂停，这表明在翻译的伸长阶段，AntISD区域具有独特的功能。实际上，最近的基因组测序数据表明，尽管核糖体RNA的抗ANTISD区域在整个原核生物中都高度保守，但许多细菌和古细菌物种并未将其用于翻译起始。有趣的是，在许多物种中，几个基因内SD位点是保守的。我假设在翻译伸长过程中，ANTISD的新功能是推动AntISD区域保护的重要因素。目的：为了了解SD诱导的暂停的广泛使用，我建议研究通过我们全基因组测量结果识别的暂停位点控制的转化过程。我的近期目标是确定抗新 - dalgarno序列在原核翻译中的更广泛作用，并确定转化暂停在蛋白质折叠，膜插入和转录后调控中的作用。这项工作将阐明所有生物包括真核生物在内的转化暂停和转化过程之间相互作用的原理，这些生物也表现出无处不在的，尽管具有未开发功能的机械机械上不同的暂停。我来自物理背景，试图通过细胞生物学和生物化学方面的实践培训来补充我的分析和光学技能。为了通过我的导师在蛋白质折叠和压力反应方面的专业知识来追求这些目标，我将获得知识库和独特的观点，以启动我自己对从机械水平到系统水平的基因表达和蛋白质合成的独立研究。