Novel Transcribing Activites in N4 Infected E. Coli

N4 感染的大肠杆菌中的新转录活动

基本信息

批准号：
7456582
负责人：
LUCIA B. B ROTHMAN-DENES
金额：
$ 49.88万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7456582
关键词：
Amino Acids Bacteriophage N4 Bacteriophages Binding Binding Proteins Biochemical Genetics Chloroplasts Code Complex Conserved Sequence Crystallization Cysteine DNA DNA Gyrase DNA Structure DNA-Directed RNA Polymerase Dissection Early Promoters Enzymes Escherichia coli Family Family member Gel Chromatography Genes Genetic Transcription Genome Grant In Vitro Infection Middle Promoters Mitochondria Modeling Molecular Weight Mutagenesis Nuclear Nucleic Acids Pathway interactions Plasmids Primer Extension Proteins Purines Recruitment Activity Research Personnel Resolution Reverse Transcriptase Polymerase Chain Reaction Role SS DNA BP Single-Stranded DNA Site Site-Directed Mutagenesis Specificity Structure Structure-Activity Relationship System Testing Transcription Initiation Transcription Initiation Site Transcriptional Activation Transcriptional Regulation Virion Work X-Ray Crystallography chemical cleavage crosslink in vivo insight member novel polypeptide programs promoter purine reconstitution scaffold sedimentation velocity transcription factor

项目摘要

DESCRIPTION (provided by applicant): Our work focuses on the structure, mechanism of promoter recognition and activation of two phage N4-coded RNA polymerases, vRNAP and N4 RNAPII, which belong to the T7 RNAP-like family. The 3,500 amino acid vRNAP recognizes a hairpin and specific sequences at its promoters. Promoter activation requires supercoiling and EcoSSB. We defined and characterized an active central domain (1,106 mini-vRNAP, the most distantly related member of the family) and have recently determined its crystal structure at 2.0 A resolution. We will define the in vivo structure of vRNAP promoters to support our model of supercoiled-induced hairpin extrusion, identify determinants of promoter recognition using biochemical and genetic approaches, determine the structure of the mini-vRNAP-promoter DNA complex by X-ray crystallography, define a nucleic acid scaffold for crystallization of the elongation complex, and define amino acid residues responsible for the EcoSSB-vRNAP interaction that elicits EcoSSB-assisted product displacement. N4 RNAPII is a heterodimer that does not recognize promoter sequences. In vivo it requires N4gp2, a ssDNA binding protein that recruits N4 RNAPII to ssDNA specifically. Middle promoters contain two sets of conserved sequences separated by 12-25 bp. We will identify all N4 RNAPII promoters and analyze their in vivo structure to test our model of promoter recognition, determine the crystal structure of N4 RNAPII and of its complex with gp2, characterize gp2 by determining its native MW, defining determinants of ssDNA-binding and of interaction with RNAPII, and the target of gp2 interaction in RNAPII. We will identify the N4-coded protein responsible for N4 RNAPII promoter specificity, and characterize its interaction with DNA, RNAPII and/or gp2 to reconstitute a system with purified components. We expect to provide new insights into strategies of promoter-RNAP interaction, into structure of factor-dependent T7-1ike RNA polymerases, and into the role of DNA structural transitions and single-stranded DNA binding proteins in transcription regulation.

描述（申请人提供）：我们的工作重点是两种噬菌体N4编码的RNA聚合酶vRNAP和N4 RNAPII的结构、启动子识别和激活机制，这两种酶属于T7 RNAP样家族。 3,500 个氨基酸的 vRNAP 可识别发夹及其启动子处的特定序列。启动子激活需要超螺旋和 EcoSSB。我们定义并表征了一个活性中心结构域（1,106 个 mini-vRNAP，该家族中关系最远的成员），并且最近以 2.0 A 分辨率确定了其晶体结构。我们将定义 vRNAP 启动子的体内结构，以支持我们的超螺旋诱导发夹挤压模型，使用生化和遗传方法确定启动子识别的决定因素，通过 X 射线晶体学确定微型 vRNAP 启动子 DNA 复合物的结构，定义了用于延伸复合物结晶的核酸支架，并定义了负责引发 EcoSSB 辅助产物置换的 EcoSSB-vRNAP 相互作用的氨基酸残基。 N4 RNAPII 是一种异二聚体，不识别启动子序列。在体内，它需要 N4gp2，一种 ssDNA 结合蛋白，可特异性地将 N4 RNAPII 招募到 ssDNA 上。中间启动子包含两组保守序列，间隔 12-25 bp。我们将鉴定所有 N4 RNAPII 启动子并分析它们的体内结构以测试我们的启动子识别模型，确定 N4 RNAPII 及其与 gp2 复合物的晶体结构，通过确定其天然 MW 来表征 gp2，定义 ssDNA 结合的决定因素和与RNAPII的相互作用，以及RNAPII中gp2相互作用的靶标。我们将鉴定负责 N4 RNAPII 启动子特异性的 N4 编码蛋白，并表征其与 DNA、RNAPII 和/或 gp2 的相互作用，以用纯化的组件重建系统。我们期望对启动子-RNAP 相互作用的策略、因子依赖性 T7-1like RNA 聚合酶的结构以及 DNA 结构转变和单链 DNA 结合蛋白在转录调控中的作用提供新的见解。