Single-molecule study of cooperation between bacterial gene expression machines

描述(申请人提供)：细胞任务由专门的大分子机器完成。这些机器中的大多数的工作原理已经被非常详细地阐明，特别是自从单分子技术出现以来。然而，对于不同大分子之间的协调机制，人们关注的却要少得多，这种协调机制经常发生在细胞内。这位候选人的长期目标是了解基本的细胞机器，特别是那些与人类疾病密切相关的机器，如何相互作用，以及它们如何协调行动，以应对环境变化。在这项提案中，候选人计划研究三种关键细菌酶之间的相互作用，即RNA聚合酶(RNAP)、核糖体和转录终止因子Rho。在第一个具体目标中，候选人将开发一种为单分子光钳实验量身定做的体外转录-翻译耦合系统。本实验将实时监测在翻译核糖体存在的情况下大肠杆菌RNAP的转录动态，这使得定量了解两个主要基因表达机制之间的相互作用成为可能。这个实验将被用作一个平台来描述不同细胞生长条件下转录-翻译偶联的动力学，并评估蛋白质辅因子和小分子抑制物在偶联中的作用。在第二个目标中，候选人将研究Rho(一种在大多数细菌中发现的通用转录终止因子)与RNAP相互作用并终止RNA合成的分子机制。他将使用高分辨率光学镊子来表征Rho作为一种马达蛋白质的机械力化学性质。有了这些知识，他将通过在单分子水平上的直接观察，进一步探索转位Rho和转录RNAP之间的相互作用。最后，在了解了RNAP和核糖体之间以及RNAP和Rho之间的偶联机制后，候选人希望探索这三种酶的协同作用，这三种酶被认为在细菌基因表达的调节中发挥关键作用。因此，阐明这些分子机器之间的协作机制可能会为抗生素的开发提供新的机遇。该项目的指导阶段将在加州大学伯克利分校的卡洛斯·布斯塔曼特博士的实验室进行。伊格纳西奥·蒂诺科博士将担任候选人的共同导师。这项研究将受益于QB3-Berkeley研究所最先进的设施和资源，以及布斯塔曼特实验室和提诺科实验室在单分子转录和翻译研究方面的专业知识。候选人希望获得额外培训的关键领域是开发单分子转录-翻译耦合系统所需的高级分子生物学和生物化学技能。候选人还计划利用授权期的指导阶段来提高他在实验室管理、教学、科学交流和撰写补助金方面的技能。这些技能将有助于他成功地过渡到独立。

DESCRIPTION (provided by applicant): Cellular tasks are accomplished by specialized macromolecular machines. The operating principles of most of these machines have been elucidated in great detail, especially since the advent of single-molecule techniques. However, much less attention has been given to the mechanism of coordination between different macromolecules, which occurs frequently inside the cell. The candidate's long-term goal is to understand how essential cellular machines, especially the ones that are closely related to human diseases, interact with one another and how they act in concert in response to environmental changes. In this proposal, the candidate plans to study the interactions between three key bacterial enzymes, namely RNA polymerase (RNAP), the ribosome, and transcription termination factor Rho. In the first specific aim, the candidate will develop an in vitro transcription-translation coupling system tailored for a single-molecule optical tweezers assay. This assay will monitor in real time the transcriptional dynamics of E. coli RNAP in the presence of a translating ribosome, which makes it possible to quantitatively understand the crosstalk between two principle gene expression machineries. This assay will then be used as a platform to characterize the dynamics of transcription- translation coupling under various cell growth conditions, and to evaluate the roles of protein cofactors and small-molecule inhibitors in the coupling. In the second aim, the candidate will investigate the molecular mechanism by which Rho, a universal transcription termination factor found in most bacteria, interacts with RNAP and terminates RNA synthesis. He will use high-resolution optical tweezers to characterize the mechanochemical properties of Rho as a motor protein. With this knowledge, he will further probe the interaction between the translocating Rho and the transcribing RNAP by direct observation at the single- molecule level. Finally, after understanding the mechanism of coupling between RNAP and the ribosome, and between RNAP and Rho, the candidate wishes to explore the concerted action of all three enzymes, which has been postulated to play a crucial role in the regulation of bacterial gene expression. Therefore elucidating the cooperative mechanism among these molecular machines may offer new opportunities for antibiotic development. The mentored phase of the project will be conducted in Dr. Carlos Bustamante's laboratory at UC Berkeley. Dr. Ignacio Tinoco will serve as the candidate's co-mentor. The research will benefit from the state- of-the-art facilities and resources at QB3-Berkeley Institute and the expertise of the Bustamante Lab and the Tinoco Lab in single-molecule transcription and translation studies. The key area in which the candidate wishes to acquire additional training is the advanced molecular biology and biochemistry skills necessary for developing the single-molecule transcription-translation coupling system. The candidate also plans to utilize the mentored phase of the award period to enhance his skills in lab management, teaching, scientific communication, and grant writing. These skills will facilitate his successful transitionto independence.