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（由适用提供）：结核分枝杆菌（MTB）引起致命的传染病，结核病，该疾病继续困扰世界人口，估计世界三分之一感染了世界。 CARD和RBPA最近被确定为直接RNA聚合酶（RNAP）结合蛋白，它们是病原体MTB中转录的必要调节剂。卡广泛分布在细菌中，包括分枝杆菌和热种类。我们的初步结果表明，卡利用一种不同的分子机制来调节转录。 RBPA是仅在细菌的放线菌家族中发现的RNAP的调节剂，包括链霉菌（SCO）和MTB。像卡一样，RBPA与其他转录调节剂没有相似之处，对于分枝杆菌的生长至关重要，并且还积极调节rRNA转录。卡和RPBA都是不寻常的转录调节剂，根据细菌转录激活剂的标准范式不起作用。我们提出了体外结构和生化方法的组合，以及在体内方法阐明通过卡和RBPA调节转录调控的分子机制。初步结果包括包含卡的热转录倡议络合物的晶体结构（分辨率为4Å）。我们建议改善这些结构结果，使用生化和生物物理方法来测试由这些结构产生的假设，并确认MTB卡通过与Thermos Card相同的机制功能。我们还获得了MTB RBPA的2.2Å分辨率晶体结构，与其RNAP靶标之一MTB SA结合在一起。我们提出了生化和生物物理方法，以测试由这种结构引起的假设，并绘制与分枝杆菌RNAP的其他相互作用。最后，我们提议结晶并确定分枝杆菌RNAP转录起始复合物的结构。