Unusual RNA polymerase from pathogenic bacterium Francisella tularensis

描述(申请人提供)：图拉氏方济氏菌，人畜共患病图拉热症的病原体，是一种高度传染性的兼性细胞内病原体。图拉氏丝虫被美国疾病控制和预防中心列为A类毒剂，具有很高的死亡率，是潜在的生物恐怖主义毒剂。最近获得的图拉氏丝虫亚种的基因组信息显示，基因组编码rpoA基因的两个平行拷贝，这两个拷贝编码不完全相同的RNA聚合酶(RNAP)？亚单位。RNAP是一种进化保守的酶，负责细菌细胞中的所有转录。到目前为止所研究的所有细菌中，是否存在相同的二聚体？亚基(？2)启动催化熟练的RNAP核心的组装(亚基组成？2？？‘)。史无前例地存在两个编码不同RNAP的基因？亚基的意思是说，在图拉氏针茅中有四种不同的？可能形成二聚体(？1)2、？1？2、？2？1和(？2)2，导致在同一细胞中存在四个不同的RNAP核心物种。因为什么？亚基通过其C-末端结构域参与启动子识别，通过序列特异性的蛋白质-DNA与启动子UP元件相互作用，并与一大套转录激活蛋白相互作用，存在两种不同的？亚基可能对图拉夫酵母基因表达的调控有深远的影响。这项建议的主要目的是确定非等同所扮演的角色？图拉氏沼虾基因表达调控的亚基。为此，我们将阐明各自的作用？RNAP组装、启动子识别和转录激活(抑制)中的亚基。为此，将通过在大肠杆菌中异源共表达或通过体外组装的方法制备所有可能的图拉氏菌RNAP核心酶的重组版本，制备相应的RNAP全酶，并利用一套鉴别的体外转录实验来确定其性质(启动子特异性、转录延伸和终止特性以及对转录调节的响应能力)。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，对高度罕见的图拉氏杆菌RNAP及其不同亚基组成的变异体的研究可能会导致开发新的图拉氏杆菌转录抑制剂和抗菌剂。 公共卫生相关性：图拉氏方济氏菌是人畜共患病图拉热症的病原体，是一种高度传染性的兼性细胞内病原体，具有不寻常的RNA聚合酶(RNAP)，它包含两个不同的α亚基。由于α亚基在RNAP组装、启动子识别和基因调控中的重要作用，对该病原菌RNAP的系统研究将揭示一种新的转录调控机制，并可能导致开发新的图拉氏杆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.