Unusual RNA polymerase from pathogenic bacterium Francisella tularensis

描述（由申请人提供）：土拉菌弗朗西斯菌是人畜共患疾病土拉菌病的病原体，是一种高度传染性的兼性细胞内病原体。土拉菌被美国疾病控制和预防中心列为a类病原体，具有高致死率，是潜在的生物恐怖主义制剂。最近可获得的土拉菌亚种基因组信息显示，基因组编码两个相邻的rpoA基因拷贝，它们编码不相同的RNA聚合酶（RNAP）。子单元。RNAP是一种进化保守的酶，负责细菌细胞的所有转录。在迄今为止研究的所有细菌中，相同的二聚体？子单元(?2)启动催化能力强的RNAP核心的组装（亚基组成？2??）两个编码不同RNAP的基因前所未有的存在？亚单位意味着在土拉菌中有四个不同的？二聚体(?1) 2、1 ?2、2 ?1、（?）2)2可能形成，导致在同一细胞中存在四个不同的RNAP核心物种。因为?亚基通过其c端结构域，通过序列特异性蛋白- dna与启动子UP元件相互作用参与启动子识别，并与大量转录激活蛋白相互作用，存在两种不同的？亚基可能对土拉菌基因表达的调控有深远的影响。本提案的主要目标是确定非同一性所扮演的角色。土拉菌基因表达调控的亚基。为此，我们将阐明每一种方法的作用。RNAP组装中的亚基，启动子识别和转录激活（抑制）。为此，将通过在大肠杆菌中异种共过表达或体外组装制备所有可能的土拉菌RNAP核心酶的重组版本，制备相应的RNAP全酶，并使用一组鉴别性体外转录试验建立其特性（启动子特异性、转录延伸和终止特性以及对转录调节剂的响应能力）。除了对了解细菌中RNAP功能和调控的基本机制具有普遍意义外，对极不寻常的土拉菌RNAP及其不同亚基组成变体的研究可能会导致开发新的土拉菌转录抑制剂和抗菌药物。

DESCRIPTION (provided by applicant): Francisella tularensis, the causative agent of the zoonotic disease tularemia, is a highly infectious facultative intracellular pathogen. F. tularensis is classified by the United States Center for Disease Control and Prevention as a category A agent, which exhibits high mortality rate and is a potential bioterrorism agent. The recent availability of genomic information for F. tularensis subspecies revealed that the genome encodes two paralogous copies of the rpoA gene, which encode non-identical RNA polymerase (RNAP) ? subunits. RNAP is an evolutionary conserved enzyme responsible for all transcription in bacterial cells. In all bacteria studied to date, a dimer of identical ? subunits (?2) initiates the assembly of the catalytically proficient RNAP core (subunit composition ?2??'). The unprecedented presence of two genes encoding different RNAP ? subunits means that in F. tularensis four different ? dimers (?1)2, ?1?2, ?2?1, and (?2)2 may form, leading to the presence of four distinct RNAP core species in the same cell. Because ? subunits, through their C-terminal domains, participate in promoter recognition by sequence-specific protein-DNA interaction with promoter UP elements and interact with a large set of transcription activation proteins, the presence of two different ? subunits may have a profound effect on the regulation of expression of genes in F. tularensis. The main goal of this proposal is to determine the roles played by non-identical ? subunits in regulation of gene expression in F. tularensis. For this purpose, we are going to elucidate the role of each ? subunit in the RNAP assembly, promoter recognition, and transcriptional activation (repression). To this end, recombinant versions of all possible F. tularensis RNAP core enzymes will be prepared by heterologous co-overexpression in E. coli or by in vitro assembly, corresponding RNAP holoenzymes will be prepared and their properties (promoter specificity, transcription elongation and termination properties and ability to respond to transcription regulators) will be established using a set of discriminative in vitro transcription assays. In addition to general significance for the understanding of basic mechanisms of RNAP function and regulation in bacteria, the proposed studies of the highly unusual F. tularensis RNAP and its variants of different subunit composition may lead to development of novel inhibitors of F. tularensis transcription and antibacterial agents. PUBLIC HEALTH RELEVANCE: Francisella tularensis, the causative agent of the zoonotic disease tularemia, a highly infectious facultative intracellular pathogen, possesses unusual RNA polymerase (RNAP), which contains two different alpha subunits. Because of important role of alpha subunit in RNAP assembly, promoter recognition and gene regulation, systematic study of RNAP from this pathogenic bacterium will uncover a novel mechanism of transcription regulation and may lead to development of new transcription inhibitors of F. tularensis and antibacterial agents.