Single-molecule study of cooperation between bacterial gene expression machines

细菌基因表达机器间协作的单分子研究

• 批准号: 8568621
• 负责人: Shixin Liu
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-15 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8568621
• 关键词: ATP phosphohydrolase; Affect; Antibiotics; Area; Attention; Award; Bacteria; Bacterial Gene Expression Regulation; Bacterial Genes; Bacterial Infections; Biochemical; Biochemistry; Biological Assay; Cells; Chemicals; Communication; Complex; Coupled; Coupling; DNA; DNA Sequence Rearrangement; DNA-Directed RNA Polymerase; Development; Dissociation; Educational process of instructing; Elongation Factor; Enzymes; Escherichia coli; Event; Fluorescence; Gene Expression; Genetic Transcription; Goals; Grant; Growth; Hybrids; In Vitro; Individual; Institutes; Kinetics; Knowledge; Laboratories; Light; Measures; Mechanics; Mentors; Messenger RNA; Modeling; Molecular; Molecular Biology; Molecular Machines; Molecular Motors; Monitor; Motion; Motor; Nature; Phase; Play; Process; Property; Protein Biosynthesis; Proteins; RNA; RNA chemical synthesis; Research; Resolution; Resources; Ribosomes; Role; Series; Speed; System; Techniques; Testing; Time; Training; Transcript; Transcription Elongation; Translating; Translations; Work; Writing; cell growth; cofactor; combinatorial; drug development; environmental change; human disease; improved; inhibitor/antagonist; insight; laser tweezer; macromolecule; operation; prevent; public health relevance; research study; response; rho; single molecule; skills; small molecule; termination factor; transcription termination

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.

描述（由申请人提供）：细胞任务由专门的大分子机器完成。大多数这些机器的工作原理已经被详细阐明，特别是自从单分子技术出现以来。然而，很少有人注意到不同大分子之间的协调机制，这经常发生在细胞内。候选人的长期目标是了解基本的细胞机器，特别是与人类疾病密切相关的细胞机器，如何相互作用，以及它们如何共同应对环境变化。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，候选人将开发一个体外转录-翻译耦合系统，用于单分子光镊分析。该检测方法将真实的实时监测E. coli RNAP中的翻译核糖体的存在下，这使得有可能定量地了解两个主要的基因表达机器之间的串扰。然后，该测定将用作表征各种细胞生长条件下转录-翻译偶联动力学的平台，并评价蛋白质辅因子和小分子抑制剂在偶联中的作用。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镊来表征Rho作为动力蛋白的机械化学特性。有了这些知识，他将通过在单分子水平上的直接观察，进一步探索易位的Rho和转录的RNAP之间的相互作用。最后，在了解RNAP和核糖体之间以及RNAP和Rho之间的耦合机制之后，候选人希望探索所有三种酶的协同作用，这三种酶被认为在细菌基因表达的调控中起着至关重要的作用。因此，阐明这些分子机器之间的协同机制可能为抗生素的开发提供新的机会。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。该研究将受益于QB 3-伯克利研究所最先进的设施和资源，以及Bustamante实验室和Tinoco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-翻译偶联系统所需的先进分子生物学和生物化学技能。候选人还计划利用奖励期间的指导阶段，以提高他在实验室管理，教学，科学交流和资助写作方面的技能。这些技能将有助于他成功地过渡到独立。