Bacterial RNAP sigma factor structure and function

描述（由申请人提供）：在细菌中，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??）与起始特异性？亚基，指导转录起始。细菌转录依赖于一级？对生存能力和可替代性都至关重要的因素是什么？控制特定规则的。控制转录起始的主要机制是通过调控？活动。戏剧性的见解来自于对？s和全酶。然而，许多挑战依然存在。在这个竞争的延续中，我们提出了进一步了解？要素结构与功能及其与辅助要素的相互作用。具体而言，我们建议：1。确定的结构基础？启动子熔化过程中与-10元素的相互作用。我们将确定复合物的晶体结构？以及体外选择的DNA适体？/-10元素相互作用对促进剂熔化至关重要。2. 绘制大肠杆菌701.1在启动子开放复合体形成的不同阶段在RNAP全酶上的位置。将使用蛋白质-蛋白质交联/映射方法（以我们的新结构1.1为指导）。基于交联的数据将与现有的基于fret的距离约束相结合，在RNAP全酶单独的背景下，以及在闭合和打开的启动子复合物中，生成？701.1的结构模型。3. 结构和功能表征？/anti-？复合物：金黄色葡萄球菌噬菌体G1 ORF67。金黄色葡萄球菌噬菌体G1 ORF67与金黄色葡萄球菌结合？结构域4，是金黄色葡萄球菌转录的有效抑制剂。我们将确定噬菌体G1 ORF67和金黄色葡萄球菌之间复合物的晶体结构。一个结构域4，并功能表征G1 ORF67抑制金黄色葡萄球菌转录的机制。4. 确定的结构基础？N与其启动子DNA相互作用。我们将确定水仙和水仙之间的晶体结构。N和启动子DNA。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及其调控的机制。我们专注于高度表征的原核RNA聚合酶，这些酶从细菌到人类的结构和功能高度保守。的吗?因子是细菌转录起始的关键调控因子。有缺陷的转录调控是许多疾病的基础。例如，许多癌蛋白是转录激活蛋白。此外，细菌RNA聚合酶已被证明是抗微生物药物的靶点，如利福平（或其衍生物），广泛用于结核病的联合治疗。对利福平耐药的细菌菌株出现的频率相当高，影响了治疗。深入了解细菌转录的机制可以为开发抗菌剂开辟新的途径。

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.