Characterization of wild-type and mutant initiation/elongation complexes in Esche

Esche 野生型和突变体起始/延伸复合物的表征

• 批准号: 7856845
• 负责人: CARLOS BUSTAMANTE
• 金额: $ 5.22万
• 依托单位: UNIVERSIDAD PERUANA CAYETANO HEREDIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7856845
• 关键词: Active Sites; Affect; Amino Acid Substitution; Animal Model; Antibiotic Therapy; Atomic Force Microscopy; Bacteria; Bacterial Genes; Bacterial Infections; Bacterial RNA; Base Pairing; Basic Science; Behavior; Biological; Biological Assay; Biomedical Research; Biotin; Capital; Cells; Characteristics; Charge; Clinical; Collaborations; Complex; Country; Coupled; Culture Media; DNA; DNA Binding; DNA Sequence; DNA Topoisomerase IV; DNA-Directed RNA Polymerase; DNA-Protein Interaction; Data; Dependence; Development; Diagnosis; Diffusion; Drug resistance; Elongation Factor; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Epidemiology; Epitopes; Escherichia coli; Event; Future; Gene Expression; Generations; Genes; Genetic; Genetic Transcription; Genome; Grant; Health; Healthcare; High Prevalence; Histidine; Homologous Gene; Hour; Image; Imagery; In Situ; Kinetics; Knowledge; Laboratories; Lead; Length; Life; Low income; Measures; Memory; Methods; Microscope; Modeling; Molecular Conformation; Molecular Motors; Monitor; Motor; Movement; Multi-Drug Resistance; Multienzyme Complexes; Mutation; Mycobacterium tuberculosis; Nucleosomes; One-Step dentin bonding system; Parents; Pathway interactions; Patients; Peru; Pharmaceutical Preparations; Physiology; Plasmids; Polymerase; Population; Positioning Attribute; Probability; Procedures; Process; Proteins; RNA; RNA Polymerase II; RNA Polymerase Inhibitor; Reaction; Regulation; Relative (related person); Reporting; Research; Resistance; Resolution; Rifampicin resistance; Rifampin; Rotation; Sigma Factor; Staging; Statistical Distributions; Streptavidin; Stress; Structural Models; System; Tail; Techniques; Testing; Time; Torque; Transcript; Transcription Elongation; Transcription Initiation; Transcription Process; Transcriptional Regulation; Translations; Tuberculosis; United States National Institutes of Health; Yeasts; abstracting; analog; arm; density; design; enzyme model; experience; grasp; histone modification; in vivo; inhibitor/antagonist; insight; laser tweezer; mutant; novel; nucleotide analog; optical traps; parent grant; promoter; public health relevance; research study; resistant strain; response; single molecule; theories; time use; tool; trait

DESCRIPTION (provided by applicant): Characterization of Wild type and mutant initiation/elongation complexes in Eschericia coli and Mycobacterium tuberculosis using Atomic Force Microscopy and Optical Tweezers "This research will be done primarily in Peru at the Universidad Peruana Cayetano Heredia in collaboration with Daniel G. Guerra, as an extension of NIH Grant No. R0137 GM 032543, active from 2008 to 2013" Abstract During the last few years we have developed novel methods and approaches to study transcription by RNA polymerase (RNAP) of Escherichia coli (Eco). Here we propose to extend these studies to emphasize several aspects of transcription initiation and elongation as the main regulation steps of gene expression and to extend the research to a medically important species, Mycobacterium tuberculosis (Mtb) and its rifampicin-resistant mutants, a health problem of high priority in Peru. We will employ an experimental platform to study the complex functioning of both Eco and Mtb enzymes in their many steps. Initiation will be directly observed by AFM imaging to chafracterize: (1) DNA binding, (2) open complex formation at specific promoters (3) open complex stability, (4) kinetics of promoter escape, (5) kinetics of sigma factor release. By using alternative promoter sequences, we will characterize the different conformational determinants that govern each one of these steps. We expect that the promoter DNA sequences, the interaction of regulators such as ppGpp and RNAP mutations will determine different subsets of conformations in the initiation complex that will solve current questions on the mechanism of transcription control. In parallel, the elongation of RNA transcripts will be observed in real-time using optical tweezers. We will measure the instantaneous velocity, processivity, pause entry, pause exit and arrest probabilities of single elongation complexes. These highly informative experimental platforms will be used to study RIF resistant mutant enzymes. By determining which transcription step is affected in the mutant enzymes, we hope to gain insight into various aspects of the in vivo translation by RIF resistant mutants. This characterization will be followed by a bottom-to-top approach using techniques such as double-mutations, culture in selective media and micro-arrays. Also, we propose to develop a platform to test and determine the mechanism of inhibition of new compounds on RNAP. Given the current absence of crystallographic Eco and Mtb structural data, single-molecule manipulation and visualization should be a powerful approach to guide the improvement of inhibitors as lead compounds for the development of new drugs. Peru, just as many other low-income countries, has a very high prevalence of TB and MDR-TB, especially concentrated in its capital, Lima. The UPCH leads highly advanced in situ biomedical research in clinical, epidemiological and genetic aspects of TB. We believe that the long term aim of controlling and defeating TB will come from the concurrence of highly advanced basic research as proposed here, development of novel drugs, and responsible healthcare strategies for management of the TB challenge. PUBLIC HEALTH RELEVANCE: Characterization of Wild type and mutant initiation/elongation complexes in Eschericia coli and Mycobacterium tuberculosis using Atomic Force Microscopy and Optical Tweezers Narrative Rifampicin is a very successful antibiotic for the treatment of tuberculosis and other bacterial infections that targets RNA polymerase (RNAP), the 5-subunit enzyme complex in charge of the regulated expression of all genes in bacteria. We will study the spatial relations and dynamics of RNA polymerases and their DNA substrates in Eschericia coli and Mycobacterium tuberculosis using single-molecule visualization and manipulation techniques to observe separately their various transcriptional intermediates. This approach constitutes a highly informative experimental platform that should make it possible to test new inhibitors of the enzymes and to guide their improvement as drug lead compounds. Moreover, since RNAP controls the expression of all bacterial genes, the study of naturally-occurring rifampicin-resistant mutants will provide insights on the development of resistant strains which could lead to future strategies to limit the emergence and spread of drug resistance.

DESCRIPTION (provided by applicant): Characterization of Wild type and mutant initiation/elongation complexes in Eschericia coli and Mycobacterium tuberculosis using Atomic Force Microscopy and Optical Tweezers "This research will be done primarily in Peru at the Universidad Peruana Cayetano Heredia in collaboration with Daniel G. Guerra, as an extension of NIH Grant No. R0137 GM 032543，从2008年到2013年，“在过去的几年中，我们开发了新的方法和方法来研究Escherichia Coli（ECO）的RNA聚合酶（RNAP）的转录。在这里，我们建议扩展这些研究，以强调转录开始和伸长的几个方面，这是基因表达的主要调节步骤，并将研究扩展到医学上重要的物种，结核分枝杆菌（MTB）及其抗rifampicin的耐药突变体，这是秘鲁高优先级的健康问题。我们将采用一个实验平台来研究ECO和MTB酶在许多步骤中的复杂功能。 AFM成像将直接观察到启动以进行造成重新分析：（1）DNA结合，（2）特定启动子处的开放复合形成（3）开放的复杂稳定性，（4）启动子逃逸的动力学，（5）Sigma因子释放的动力学。通过使用替代启动子序列，我们将表征控制这些步骤中每个步骤的不同构象决定因素。我们期望启动子DNA序列，PPGPP和RNAP突变等调节剂的相互作用将确定起始复合物中不同的构象子集，这将解决有关转录控制机理的当前问题。同时，将使用光学镊子实时观察RNA转录物的伸长。我们将衡量单个伸长式复合物的瞬时速度，加工性，暂停输出，停顿出口和停滞概率。这些高度信息丰富的实验平台将用于研究抗性突变酶。通过确定突变酶中哪个转录步骤受到影响，我们希望能够深入了解抗rif抗性突变体体内翻译的各个方面。这种表征将采用诸如双重突变，选择性媒体和微阵列中的文化之类的技术。另外，我们建议开发一个平台来测试和确定抑制RNAP上新化合物的机制。鉴于当前缺乏晶体学生态和MTB结构数据，单分子的操作和可视化应该是一种有力的方法，可以指导抑制剂作为开发新药的铅化合物的改善。 与许多其他低收入国家一样，秘鲁的结核病和MDR-TB的患病率很高，尤其是集中在其首都利马。 UPCH领导着TB的临床，流行病学和遗传方面高度先进的原位生物医学研究。我们认为，控制和击败结核病的长期​​目标将来自此处提出的高级基础研究，新型药物的发展以及负责任的医疗保健策略，以管理结核病挑战。 公共卫生相关性：使用原子力显微镜和光学镊子叙事rifampicin在大肠杆菌和结核分枝杆菌中野生型和突变的起始/伸长络合物的表征是一种非常成功的抗生素，可用于治疗结核病和其他细菌感染，可用于RNA Polemase（RNA Polemase）（RNA Polymase in of RNA Polymase（RNA Polymase））（RNA Polymase in of Rna Polymerase（RNA Polymase）（RNA Polimase）5-rnap），rnape in 5调节细菌中所有基因的表达。我们将使用单分子可视化和操纵技术研究RNA聚合酶及其DNA底物的空间关系和动力学，以分别观察其各种转录中间体。这种方法构成了一个非常有用的实验平台，该平台应该可以测试酶的新抑制剂并指导其作为药物铅化合物的改进。此外，由于RNAP控制了所有细菌基因的表达，因此对自然存在的利福平耐药突变体的研究将提供有关抗性菌株发展的见解，这可能导致未来的策略限制耐药性的出现和传播。