Inter and intramolecular communications in transcription

描述(申请人提供)：转录是调节细胞功能的中心点之一。了解转录的分子机制及其调控仍然是生物学中最重要的目标之一。DNA依赖的RNA聚合酶(RNAP)是一种能够将基因序列中编码的信息复制到mRNA产物中的酶，在转录过程中起着至关重要的作用。我们的研究集中在细菌和古生菌的RNAP。这些酶是最简单的多亚单位聚合酶之一，为获得RNAP活性的详细分子理解提供了最好的机会。我们的长期目标是了解RNA聚合酶启动转录的分子机制。细菌RNAP是一种重要的酶，是新抗生素的靶标。详细了解细菌酶所特有的RNAP特性将有助于开发针对RNAP的新抗生素。现在很明显，转录启动涉及许多RNAP-启动子接触之间的复杂相互作用。虽然我们开始了解RNAP如何在转录起始反应中利用这些接触，但在全面了解转录起始之前，需要填补许多知识空白。在这个项目中，我们将重点填补其中的三个空白。在目标1中，我们将确定保守的非模板-7胸腺嘧啶在细菌RNA聚合酶熔化启动子中的作用。在目标2中，我们将确定-35保守启动子元件和连接-10和-35启动子元件的间隔区在细菌RNA聚合酶熔化启动子中的作用。在目标3中，我们将研究RNAP与核心启动子元件和第二启动子元件在细菌RNA聚合酶的启动子逃逸中的相互作用。此外，我们还将开始研究古生菌RNA聚合酶(AIM 4)启动转录的机制。古生菌RNA聚合酶表现出真核生物样的结构，但细菌样的启动子正在熔化(即熔化不需要依赖于ATP的辅助活性)。因此，对古生菌聚合酶的研究将有助于深入了解转录起始机制的进化，并有助于理解这一过程的一般机制特征。在我们的研究中，我们将结合使用荧光技术以及对启动子熔化反应的诱变、动力学、热力学和结构分析。在完成这些研究后，将获得对基本生物过程的机械性见解。公共卫生相关性：该项目将为细菌RNA聚合酶启动转录的机制提供新的见解。这些信息将使针对细菌RNA聚合酶的新抗生素的开发成为可能。针对新的靶点开发新的抗生素对于对抗新的抗生素耐药菌株至关重要。

DESCRIPTION (provided by applicant): Transcription is one of the central points of regulation of cellular functions. Understanding molecular mechanisms of transcription and its regulation remains one of the most important goals in biology. DNA-dependent RNA polymerases (RNAP), the enzymes capable of faithfully copying the information encoded in gene sequence into the mRNA product, are of central importance in the process of transcription. Our research is focused on bacterial and archaeal RNAP's. These enzymes are among the simplest multisubunit polymerases, offering the best chance for obtaining detailed molecular understanding of RNAP activity. Our long term goal is to understand the molecular mechanism by which RNA polymerase initiates transcription. Bacterial RNAP is an attractive target for new antibiotics since it is an essential enzyme. Detailed understanding of RNAP properties unique to the bacterial enzyme will aid the development of new antibiotics targeting RNAP. It is clear now that transcription initiation involves a complex interplay between numerous RNAP-promoter contacts. While we are beginning to understand how RNAP utilizes these contacts in transcription initiation reaction, many gaps in knowledge will need to be filled before a comprehensive understanding of transcription initiation is achieved. In this project we will focus on filling in three of such gaps. In aim 1 we will determine the role of conserved nontemplate -7 thymine in promoter melting by bacterial RNA polymerase. In aim 2 we will determine the role of -35 conserved promoter element and the spacer connecting -10 and -35 promoter elements in promoter melting by bacterial RNA polymerase. In aim 3, we will investigate the interplay between RNAP interactions with the core and secondary promoter elements in promoter escape by bacterial RNA polymerase. Furthermore, we will also begin to investigate the mechanism of transcription initiation by archaeal RNA polymerase (aim 4). Archaeal RNA polymerase exhibits eukaryote-like structure but bacteria-like promoter melting (i.e. no auxiliary ATP-dependent activities are necessary for melting). Thus, the studies with archaeal polymerase will provide insights into the evolution of transcription initiation mechanism and will facilitate understanding of general mechanistic features this process. In our studies we will utilize a combination of fluorescence techniques together with mutagenesis, kinetic, thermodynamic and structural analysis of promoter melting reactions. Upon completion of these studies mechanistic insights to a fundamental biological process will be gained. PUBLIC HEALTH RELEVANCE: This project will provide new insights into the mechanism of transcription initiation by bacterial RNA polymerase. Such information will enable development of new antibiotics targeting bacterial RNA polymerase. Development of new antibiotics targeting new targets is essential to combating emerging antibiotic-resistant strains.