Structure/function analyses of essential mycobacterial transcription regulators

分枝杆菌必需转录调节因子的结构/功能分析

• 批准号: 9041636
• 负责人: ELIZABETH A CAMPBELL
• 金额: $ 40万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9041636
• 关键词: Actinomyces Infections; Affinity; Bacteria; Binding; Binding Proteins; Biochemical; ChIP-seq; Collaborations; Communicable Diseases; Complex; DNA; DNA Damage; DNA-Directed RNA Polymerase; Enzymes; Family; Genetic Transcription; Genome; Genus Mycobacterium; Growth; Health; Holoenzymes; In Vitro; Infection; Institutes; Life; Literature; Malignant Neoplasms; Maps; Modeling; Molecular; Mus; Mycobacterium tuberculosis; Oxidative Stress; Plague; Population; Regulation; Resolution; Ribosomal RNA; Roentgen Rays; Role; Starvation; Streptomyces coelicolor; Structure; Testing; Thermus; Transcription Coactivator; Transcription Initiation; Transcriptional Regulation; Tuberculosis; antimicrobial; base; biophysical techniques; design; improved; in vivo; interest; killings; mycobacterial; novel therapeutics; pathogen; promoter; transcription factor

 DESCRIPTION (provided by applicant): Mycobacteria tuberculosis (Mtb) causes the deadly infectious disease, tuberculosis, which continues to plague the world's population, with an estimated one third of the world infected. CarD and RbpA have recently been identified as direct RNA polymerase (RNAP) binding proteins that are essential regulators of transcription in the pathogen Mtb. CarD is widely distributed among bacteria, including Mycobacterium and Thermus species. Our preliminary results indicate that CarD utilizes a distinct molecular mechanism for regulating transcription. RbpA is a regulator of RNAP found only in the actinomycete family of bacteria, including Streptomyces coelicolor (Sco) and Mtb. Like CarD, RbpA has no similarity to other transcription regulators, is essential for growth in mycobacteria and also positively regulates rRNA transcription. Both CarD and RpbA are unusual transcription regulators that do not function according to the standard paradigm for bacterial transcription activators. We propose a combination of in vitro structural and biochemical approaches, and in vivo approaches to elucidate the molecular mechanisms for transcription regulation by CarD and RbpA. Preliminary results include crystal structures of thermus transcription initiation complexes containing CarD (4 Å-resolution). We propose to improve on these structural results, use biochemical and biophysical approaches to test hypotheses that arise from these structures as well as to confirm that Mtb CarD functions through the same mechanism as thermus CarD. We have also obtained a 2.2 Å-resolution crystal structure of Mtb RbpA bound to one of its RNAP targets, the Mtb sA. We propose biochemical and biophysical approaches to test hypotheses that arise from this structure and to map additional interactions with the mycobacterial RNAP. Finally, we propose to crystallize and determine the structure of mycobacterial RNAP transcription initiation complexes.

 描述（由申请人提供）：结核分枝杆菌 (Mtb) 引起致命的传染病结核病，该疾病继续困扰着世界人口，估计世界上三分之一的人受到感染。 CarD 和 RbpA 最近被鉴定为直接 RNA 聚合酶 (RNAP) 结合蛋白，是病原体 Mtb 转录的重要调节因子。 CarD 广泛分布于细菌中，包括分枝杆菌和栖热菌属。我们的初步结果表明 CarD 利用独特的分子机制来调节转录。 RbpA 是仅在放线菌家族细菌中发现的 RNAP 调节因子，包括天蓝色链霉菌 (Sco) 和 Mtb。与 CarD 一样，RbpA 与其他转录调节因子没有相似之处，对于分枝杆菌的生长至关重要，并且还积极调节 rRNA 转录。 CarD 和 RpbA 都是不寻常的转录调节因子，它们不按照细菌转录激活因子的标准模式发挥作用。我们提出结合体外结构和生化方法以及体内方法来阐明 CarD 和 RbpA 转录调控的分子机制。 初步结果包括含有 CarD 的栖热菌转录起始复合物的晶体结构（4 Å 分辨率）。我们建议改进这些结构结果，使用生物化学和生物物理方法来测试由这些结构产生的假设，并确认 Mtb CarD 通过与 thermus CarD 相同的机制发挥作用。 我们还获得了 Mtb RbpA 与其 RNAP 靶点之一 Mtb sA 结合的 2.2 Å 分辨率晶体结构。我们提出了生物化学和生物物理方法来测试由该结构产生的假设，并绘制与分枝杆菌 RNAP 的额外相互作用。最后，我们建议结晶并确定分枝杆菌 RNAP 转录起始复合物的结构。