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: 8212394
• 负责人: Katsuhiko Murakami
• 金额: $ 27.85万
• 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/8212394
• 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）：本提案的目的是确定古细菌RNA聚合酶及其与辅助蛋白因子和核酸的复合物的X射线晶体结构。在真核生物中的转录装置可以被描述为其真核RNA聚合酶II（Pol II）对应物的简化版本，包括Pol II样RNA聚合酶和通用转录因子TBP、TFB、TFE和TFS（分别为真核TBP、TFIIB、TFIIE 1和TFS直向同源物）。值得注意的是，在古细菌基因组中发现的转录调节因子与细菌因子密切相关。因此，阐明的转录机制，这是一个马赛克的细菌和真核生物的功能，将提供一个基础，统一的见解，从细菌，古细菌和真核生物系统的基本转录机制在所有三个领域的生活。我们最近报道的第一个X射线晶体结构的古细菌RNA聚合酶硫磺硫化叶菌在3.4 E分辨率。这是我们工作中的一个重大突破，并为使用下文所述的结构生物学方法来解决关于Escherichia中转录机制的问题提供了基础。1.完成硫磺硫化叶菌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提高到大于2.5 E的分辨率，以便精确地比较细菌、古细菌和真核生物RNA聚合酶的结构。2.阐明DNA开放和转录起始的机制。古细菌通用转录因子TFB和TFE与RNA聚合酶形成稳定的复合物。TFB在将RNA聚合酶募集到DNA启动子和转录起始中起关键作用。TFE促进双链DNA启动子的打开。为了理解DNA打开和转录起始的机制，我们建议确定古细菌RNA聚合酶与TFB和与TFE的复合物的X射线晶体结构，以及整个RNA聚合酶转录前起始复合物和与TBP-TFB-启动子DNA的打开复合物。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聚合酶与DNA之间的相互作用。由于古细菌RNA聚合酶与酵母Pol II之间的高度结构相似性，我们将通过基于古细菌开放复合体结构的突变分析来研究酵母中转录起始位点选择的机制。 公共卫生相关性：基因表达是所有细胞生物的基础，对于理解细胞发育、维持和疾病至关重要。古细菌的转录系统适合于X射线晶体学研究，而且，与真核细胞中的转录系统相似。因此，拟议的研究将提供一个结构框架，分析四十年的转录研究在不同的真核系统，也提供了许多有用的见解多亚基聚合酶在生命的三个领域的进化。