Probing the function of translational pausing in bacterial protein synthesis

项目总结/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 新生链的降解。直到3年前，我们才第一次对全球的速度有了看法。 翻译与体内子密码子解析。使能技术是核糖体分析-深度测序 核糖体保护的mRNA片段-在加州大学旧金山分校的韦斯曼实验室开发。通过结合核糖体 用计算方法分析，我现在已经开始努力破译翻译停顿是如何发生的， 调节蛋白质合成。 自从在加州大学旧金山分校开始工作以来，我有了一个令人惊讶的发现， 细菌发生在内部的Shine-Dalgarno（SD）序列，由它们与抗Shine-Dalgarno（SD）序列的相互作用驱动。 延伸核糖体的Dalgarno（antiSD）区域。目前的模式，建立了闪耀和 Dalgarno在1975年提出的一个新的观点是，核糖体抗SD区的主要作用是定义翻译起始位点， 原核生物我的发现是，有保守的和无处不在的暂停在内部SD网站建议一个独特的 在翻译的延伸阶段为antiSD区域起作用。事实上，最近的基因组测序 数据显示，尽管核糖体RNA的antiSD区域在整个 在原核生物中，许多细菌和古细菌物种不使用它来启动翻译。有趣的是，几个 基因内SD位点在许多物种中是保守的。我推测，抗SD的这种新功能， 翻译延伸是驱动抗SD区保守性的重要因素。 目的：为了了解广泛使用的SD诱导暂停，我建议调查的共同点， 翻译过程由我们的全基因组测量确定的暂停位点控制。我 近期目标是确定抗Shine-Dalgarno序列在原核翻译中的更广泛作用， 以确定翻译暂停在蛋白质折叠，膜插入和转录后的作用， 调控本文将阐明翻译停顿和合作停顿之间相互作用的原则。 翻译过程中的所有生物体，包括真核生物，也表现出普遍存在的，虽然 机械性的不同停顿，具有未探索的功能。 来自物理学背景，我正在寻求补充我的分析和光学技能， 在细胞生物学和生物化学方面扎实的实践训练。在追求这些目标与我的导师的专业知识， 蛋白质折叠和应激反应，我将获得知识基础和独特的视角， 我将从机制层面对基因表达和蛋白质合成进行独立研究 到系统层面。

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.