Probing the function of translational pausing in bacterial protein synthesis

描述（由申请人提供）：蛋白质合成的速度可以影响所有共翻译过程，从新生链的折叠到降解。直到3年后， 之前，我们有了第一个关于体内子密码子解析翻译速度的全局视图。这项技术是由加州大学旧金山分校的韦斯曼实验室开发的核糖体分析--核糖体保护的mRNA片段的深度测序。通过将核糖体分析与计算方法相结合，我现在已经开始努力破译翻译暂停如何调节蛋白质合成。 自从在加州大学旧金山分校开始，我令人惊讶地发现，细菌中的大多数翻译暂停位点发生在内部Shine-Dalgarno（SD）序列上，由它们与延长核糖体的anti-Shine-Dalgarno（antiSD）区域的相互作用驱动。Shine和Dalgarno在1975年建立的当前范式是，核糖体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，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功能antiSD区域在翻译的延伸阶段。事实上，最近的基因组测序数据显示，尽管核糖体RNA的antiSD区域在整个原核生物中高度保守，但许多细菌和古细菌物种并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，这种新的功能antiSD在翻译延伸是一个重要因素驱动的antiSD区域的保护。目的：为了理解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更广泛的作用，在原核翻译的anti-Shine-Dalgarno序列，并确定翻译暂停蛋白质折叠，膜插入和转录后调控的作用。这项工作将阐明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，真核生物也表现出普遍存在的，尽管机制上不同的暂停，但功能尚未探索。 来自物理学的背景，我正在寻求补充我的分析和光学技能与坚实的动手训练细胞生物学和生物化学。在追求这些目标与我的导师在蛋白质折叠和应激反应的专业知识，我将获得知识基础和独特的视角，从机制层面到系统层面启动我自己的基因表达和蛋白质合成的独立调查。

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.