Bacterial RNAP sigma factor structure and function

描述(申请人提供)：细菌中的450 kDa RNA聚合酶(RNAP)全酶，由进化上保守的催化核心(亚基组成？2？)组成。结合特定的入门仪式？亚基，指导转录的启动。细菌转录依赖于初级基因吗？对生存至关重要的因素，以及控制特定调控的替代方案？S说。控制转录启动的一个主要机制是通过调节？活动。S和全酶的结构研究带来了戏剧性的见解。然而，许多挑战依然存在。在这一相互竞争的继续中，我们建议进行研究，以加深我们对？因素的结构和功能，以及与辅助因素的相互作用。具体地说，我们建议：1.确定结构基础？启动子熔化启动过程中与-10元件的相互作用。我们将确定配合物的晶体结构？在体外选择的DNA适配子模拟对启动子熔化至关重要的？/-10元素相互作用。2.在启动子开放复合体形成的不同阶段定位E.Coli？701.1在RNAP全酶上的位置。将使用蛋白质-蛋白质交联/作图方法(在我们1.1的新结构的指导下)。基于交联的数据将与现有的基于FRET的距离约束相结合，以生成在仅在全酶以及在封闭和开放启动子复合体中的RNAP全酶的背景下？701.1的结构模型。3.从结构和功能上表征？/反？复合体：金黄色葡萄球菌噬菌体G1 ORF67。金黄色葡萄球菌噬菌体G1 ORF67与金黄色葡萄球菌A区4结合，是金黄色葡萄球菌转录的有效抑制因子。我们将确定噬菌体G1ORF67与金黄色葡萄球菌A结构域4之间的复合物的晶体结构，并从功能上表征G1ORF67抑制金黄色葡萄球菌转录的机制。4.确定N与其启动子DNA相互作用的结构基础。我们将确定Aquifex aeolicus？N与启动子DNA之间的复合体的晶体结构。公共卫生相关性：转录是基因表达的主要控制点，RNA聚合酶是转录的中心酶。我们的长期目标是了解转录的机制及其调控。我们关注的是高度特征化的原核生物RNA聚合酶，它从细菌到人类都具有高度保守的结构和功能。那个？因子是细菌转录启动的关键调控因子。转录调控缺陷是许多疾病的基础。例如，许多癌蛋白是转录激活蛋白。此外，细菌RNA聚合酶是广泛用于治疗结核病的联合疗法中广泛使用的抗菌剂，如利福平(或其衍生物)的已被证实的靶点。对利福平耐药的细菌菌株以相当高的频率出现，影响了治疗。对细菌转录机制的深入了解可以为抗微生物药物的开发开辟新的途径。

DESCRIPTION (provided by applicant): In bacteria, the 450 kDa RNA polymerase (RNAP) holoenzyme, comprising the evolutionarily conserved catalytic core (subunit composition ?2??'?) combined with the initiation-specific ? subunit, directs transcription initiation. Bacterial transcription depends on a primary ? factor that is essential for viability, as well as alternative ?'s that control specific regulons. A major mechanism to control transcription initiation is through regulation of ? activity. Dramatic insights have come from structural studies of ?'s and holoenzymes. Nevertheless, many challenges remain. In this competing continuation, we propose studies to further our understanding of ? factor structure and function, and interactions with accessory factors. Specifically, we propose to: 1. Determine the structural basis for ? interactions with the -10 element in the initiation of promoter melting. We will determine crystal structures of complexes between ? and in vitro-selected DNA aptamers that mimic ?/-10 element interactions critical for promoter melting. 2. Map the position of E. coli ?701.1 on the RNAP holoenzyme during different stages of promoter open complex formation. A protein-protein crosslinking/mapping approach (guided by our new structure of ?1.1) will be used. The crosslinking-based data will be combined with existing FRET-based distance constraints to generate structural models of ?701.1 in the context of the RNAP holoenzyme in holoenzyme alone, as well as in closed and open promoter complexes. 3. Structurally and functionally characterize ?/anti-? complexes: Staphylococcal aureus phage G1 ORF67. S. aureus phage G1 ORF67 binds to S. aureus ?A domain 4 and is a potent inhibitor of S. aureus transcription. We will determine crystal structures of complexes between phage G1 ORF67 and S. aureus ?A domain 4, and functionally characterize the mechanism of G1 ORF67 inhibition of S. aureus transcription. 4. Determine the structural basis for ?N interactions with its promoter DNA. We will determine the crystal structure of a complex between Aquifex aeolicus ?N and promoter DNA. PUBLIC HEALTH RELEVANCE: Transcription is the major control point of gene expression and RNA polymerase is the central enzyme of transcription. Our long term goal is to understand the mechanism of transcription and its regulation. We focus on highly characterized prokaryotic RNA polymerases, which have a high degree of conservation of structure and function from bacteria to man. The ? factors are the key regulators of bacterial transcription initiation. Defective transcription regulation underlies many disorders. For example, many oncoproteins are transcriptional activator proteins. Moreover, the bacterial RNA polymerase is a proven target for antimicrobials, such as rifampicin (or its derivatives), widely used in combination therapy to treat tuberculosis. Bacterial strains resistant to rifampicin arise with appreciable frequency, compromising treatment. Insights into the mechanism of bacterial transcription can lead to new avenues for the development of antimicrobials.