X-ray crystallographic studies of multi-subunit nucleic acid polymerases

多亚基核酸聚合酶的 X 射线晶体学研究

• 批准号: 7781374
• 负责人: Katsuhiko Murakami
• 金额: $ 28.17万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7781374
• 关键词: Address; Applications Grants; Archaea; Archaeal Genome; Archaeal RNA; Bacteria; Bacteriophage N4; Biochemical; Biochemistry; Biological Models; Cells; Chicago; Collaborations; Complex; Crystallization; DNA; DNA-Directed RNA Polymerase; Data; Data Collection; Development; Disease; Enzymes; Eukaryotic Cell; Evolution; Fingers; Fluorescence Spectroscopy; Foundations; Funding; Gene Expression; General Transcription Factors; Genetic; Genetic Code; Genetic Transcription; Goals; Grant; Home environment; Knowledge; Laboratories; Life; Maintenance; Maps; Modeling; Molecular; National Institute of General Medical Sciences; Nucleic Acids; Organism; Orthologous Gene; Phase; Play; Polymerase; Positioning Attribute; Process; Proteins; R-factor; RNA Polymerase II; Recruitment Activity; Reporting; Research; Resolution; Roentgen Rays; Role; Saints; Source; Structure; Sulfolobus solfataricus; Synchrotrons; System; TATA-Box Binding Protein; Transcription Factor TFIIB; Transcription Initiation; Transcription Initiation Site; Trees; United States National Institutes of Health; Universities; Work; X-Ray Crystallography; Yeasts; base; beamline; ds-DNA; electron density; improved; insight; interest; polypeptide; promoter; public health relevance; resistance factors; structural biology

DESCRIPTION (provided by applicant): The goal of this proposal is to determine the X-ray crystal structures of archaeal RNA polymerase and its complexes with auxiliary protein factors and nucleic acid. The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase II (Pol II) counterpart, comprising a Pol II-like RNA polymerase and the general transcription factors TBP, TFB, TFE and TFS (eukaryotic TBP, TFIIB, TFIIE1 and TFS orthologs, respectively). Remarkably, the transcription regulators found in archaeal genomes are closely related to bacterial factors. Therefore, elucidating the transcription mechanism in Archaea, which is a mosaic of bacterial and eukaryotic features, would provide a foundation for unifying insights from bacterial, archaeal and eukaryotic systems into basic transcription mechanisms across all three domains of life. We recently reported the first X-ray crystal structure of the archaeal RNA polymerase from Sulfolobus solfataricus at 3.4 E resolution. This represents a major breakthrough in our work and provides the foundation for addressing questions on the transcription mechanism in Archaea using structural biology approaches as described below. 1. Complete the Refinement of the Sulfolobus solfataricus RNA Polymerase Model. Our goal is to increase the resolution of the archaeal RNA polymerase structure from 3.4 E to greater than 2.5 E resolution in order to precisely compare the structures of bacterial, archaeal and eukaryotic RNA polymerases. 2. Elucidate the Mechanism of DNA Opening and Transcription Initiation. The archaeal general transcription factors TFB and TFE form stable complexes with RNA polymerase. TFB plays critical roles in recruiting RNA polymerase to the DNA promoter and transcription initiation. TFE facilitates opening of the double-stranded DNA promoter. To understand the mechanism of DNA opening and transcription initiation, we propose to determine the X-ray crystal structures of the archaeal RNA polymerase in a complex with TFB and with TFE, and the entire RNA polymerase transcription pre- initiation complex and open complex with TBP-TFB-promoter DNA. We will carry out structure-based mutational analysis to elucidate the interaction between RNA polymerase and TFB/TFE as well as RNA polymerase and DNA. Because of the high structure similarity between archaeal RNA polymerase and yeast Pol II, we will investigate the mechanism of transcription start site selection in yeast by using mutational analysis based on the archaeal open complex structure. PUBLIC HEALTH RELEVANCE: Gene expression is fundamental to all cellular organisms and is critical for understanding cell development, maintenance, and disease. The archaeal transcription system is amenable to X-ray crystallographic studies, and moreover, is similar to that in eukaryotic cells. Therefore, the proposed studies will provide a structural framework for analyzing four decades of transcription research on diverse eukaryotic systems, and also provide many useful insights into evolution of multi-subunit polymerases in the three domains of life.

描述（由申请人提供）：该建议的目的是确定古细菌RNA聚合酶的X射线晶体结构及其具有辅助蛋白质因子和核酸的复合物。古细菌中的转录设备可以描述为其真核RNA聚合酶II（POL II）的简化版本，包括POL II样RNA聚合酶和一般转录因子TBP，TFB，TFE和TFS（真核生物TBP，TBP，TFIIB，TFIIB，TFIIEIE1和TFS或TFS或TFS），分别是相应的。值得注意的是，古细胞基因组中发现的转录调节剂与细菌因子密切相关。因此，阐明古细菌中的转录机制，这是细菌和真核特征的镶嵌物，将为将细菌，古细菌和真核系统从所有三个生命领域的基本转录机制中统一见解提供基础。我们最近报道了以3.4 e分辨率的溶质溶液中的古细菌RNA聚合酶的第一个X射线晶体结构。这是我们工作中的一个重大突破，并为解决古细菌中的转录机制的问题提供了基础，如下所述，结构生物学方法。 1。完成磺胺溶液RNA聚合酶模型的细化。我们的目标是将古细菌RNA聚合酶结构的分辨率从3.4 E增加到大于2.5 E的分辨率，以精确地比较细菌，古细菌和真核RNA聚合酶的结构。 2。阐明DNA开放和转录启动的机理。古细胞一般转录因子TFB和TFE与RNA聚合酶形成稳定的复合物。 TFB在募集RNA聚合酶至DNA启动子和转录启动方面起关键作用。 TFE促进了双链DNA启动子的开放。为了了解DNA开放和转录起始的机制，我们建议确定与TFB和TFE的复合物中古细胞RNA聚合酶的X射线晶体结构，以及与TBP-TFB-TFB启动器DNA的整个RNA聚合酶转录启动复合物和开放式复合物。我们将进行基于结构的突变分析，以阐明RNA聚合酶与TFB/TFE以及RNA聚合酶和DNA之间的相互作用。由于古细菌RNA聚合酶和酵母菌II之间的结构相似性很高，我们将通过基于古细胞开放的复杂结构的突变分析来研究酵母中转录起始位点选择的机理。 公共卫生相关性：基因表达对所有细胞生物都是基础的，对于理解细胞发育，维持和疾病至关重要。古细胞转录系统适合X射线晶体学研究，此外，与真核细胞相似。因此，拟议的研究将提供一个结构框架，用于分析有关多种真核系统的四十年转录研究，并为在生命的三个领域中多生物共聚合酶的演变提供了许多有用的见解。