Regulation of elongation by RNA polymerase and ribosome via intrinsic signals and transcription-translation coupling

了解控制细胞中基因表达的分子机制是开发有害和有益生物体的智能操作策略，开发新的抗菌和抗真菌药物的重要组成部分，重要的是，对于理解生命的出现和进化。在细菌中，基因表达的两个步骤，将基因复制到RNA（转录）和在RNA上生产蛋白质（翻译）是耦合的。这意味着这两种分子机制的紧密相互作用和相互调节，这决定了基因表达的总体速度，这反过来对所有细胞过程都很重要。这种调控和决定转录和翻译速率的机制尚不清楚。我们是第一个开发出独特实验系统的实验室，在这个系统中，基因表达的两个步骤都可以从纯化的组分组装起来，并在试管中同时进行研究。我们还拥有一些独特的生化技术，例如，这使我们能够描述参与调节HIV-1发展的机制，并揭示抗生素Tagetitoxin的作用模式。在本提案中，通过使用这些技术，我们将研究决定基因表达两个步骤的速率的因素：(i)我们将采用系统的方法来理解DNA中可能影响转录速率的信号；（ii）我们将研究转录和翻译的相互作用和相互调节。复杂的转录和翻译调控是病原菌存活的主要因素。了解这两个基因表达步骤之间的关系将提高我们对这些细菌的认识，从而为对抗传染病提供新的工具。我们与一家生物技术公司合作，应用我们在研究中开发的实验工具来阐明新的抗生素，RNA聚合酶抑制剂的作用模式。由于处于转录和翻译的交界，本研究将对这两个广阔的分子生物学领域都有价值。我们与英国领先的实验室合作，他们将使用本研究中设计的工具，通过生物物理方法（如单分子技术、晶体学、冷冻电子显微镜）研究基因表达的机制。此外，在试管中同时组装两种主要的细胞机器可能成为生产人工组装生物体的第一步。

Understanding the molecular mechanisms governing the expression of genes in the cell is an essential part of developing strategies for intelligent manipulation of harmful and useful organisms, development of new antimicrobial and antifungal agents, and, importantly, for understanding emergence and evolution of Life.In bacteria, both steps of gene expression, copying genes into RNA (transcription) and production of proteins on RNA (translation), are coupled. This implies tight interactions and mutual regulation of these two molecular machineries, which determine the overall rate of gene expression, which, in turn, is important for all cellular processes. This regulation and mechanisms determining the rates of transcription and translation are poorly understood. We are the first laboratory who developed a unique experimental system where both steps of gene expression can be assembled from purified components and investigated simultaneously in the test tube. We also possess some unique biochemical techniques, which allowed us for example to describe the mechanism involved in regulation of the development of HIV-1, and to uncover mode of action of antibiotic Tagetitoxin. In this proposal, by using these techniques, we will investigate what factors determine rates of both steps of gene expression: (i) we will apply a systematic approach to understand the signals in the DNA that may influence the rate of transcription; (ii) we will investigate the interactions and mutual regulation of transcription and translation. Sophisticated regulation of transcription and translation are the major factors for survival of pathogenic bacteria. Understanding of relations between these two steps of gene expression will improve our knowledge of these bacteria, and thus provide new tools for fighting infectious diseases. We collaborate with a bio-tech company on applying experimental tools developed in our research to elucidation of modes of action of new antibiotics, inhibitors of RNA polymerase. Being at the interface of transcription and translation, the proposed study will be valuable for both of these vast fields of molecular biology. We collaborate with the leading UK laboratories who will use the tools designed in this study to investigate mechanisms of gene expression by biophysical methods, such as single-molecule techniques, crystallography, cryo-electron microscopy. Furthermore, simultaneous assembly of the two major cellular machineries in the test tube may become the first step for production of artificially assembled living organisms.