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

基本信息

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
项目类别：
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

项目摘要

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）：本提案的目标是确定古菌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。古细菌中的转录装置可以被描述为真核 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（真核 TBP、TFIB、分别为 TFIIE1 和 TFS 直向同源物）。值得注意的是，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，阐明古细菌（细菌和真核特征的镶嵌体）的转录机制，将为将细菌、古细菌和真核系统的见解统一到生命所有三个领域的基本转录机制提供基础。我们最近报道了来自硫磺硫化叶菌的古菌 RNA 聚合酶的第一个 X 射线晶体结构，分辨率为 3.4 E。这代表了我们工作的重大突破，并为使用结构生物学方法解决古细菌转录机制问题提供了基础，如下所述。 1. 完成硫磺硫化叶菌RNA聚合酶模型的完善。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 分辨率，以便精确比较细菌、古细菌和真核 RNA 聚合酶的结构。 2. 阐明DNA开放和转录起始的机制。古菌通用转录因子 TFB 和 TFE 与 RNA 聚合酶形成稳定的复合物。 TFB 在将 RNA 聚合酶招募到 DNA 启动子和转录起始中发挥着关键作用。 TFE 促进双链 DNA 启动子的打开。为了了解DNA打开和转录起始的机制，我们建议确定古菌RNA聚合酶与TFB和TFE复合物的X射线晶体结构，以及整个RNA聚合酶转录预起始复合物和与TBP的开放复合物-TFB-启动子DNA。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。由于古菌RNA聚合酶与酵母Pol II结构高度相似，我们将通过基于古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体的基础，对于了解细胞发育、维持和疾病至关重要。古菌转录系统适合 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，拟议的研究将为分析四十年来对不同真核系统的转录研究提供一个结构框架，并为生命三个领域中多亚基聚合酶的进化提供许多有用的见解。