Novel Transcribing Activites in N4 Infected E. Coli

N4 感染的大肠杆菌中的新转录活动

• 批准号: 7081315
• 负责人: LUCIA B. B ROTHMAN-DENES
• 金额: $ 49.84万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 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/7081315
• 关键词: DNA; DNA binding protein; DNA directed RNA polymerase; DNA footprinting; Escherichia coli; X ray crystallography; atomic force microscopy; bacterial genetics; coliphages; crosslink; enzyme activity; enzyme complex; enzyme structure; fluorescence resonance energy transfer; gene mutation; genetic promoter element; genetic transcription; intermolecular interaction; nucleic acid sequence; nucleic acid structure; protein structure function; site directed mutagenesis; virus genetics; virus protein

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（由申请人提供）：我们的工作着重于启动子识别的结构，机理以及属于T7 RNAP样家族的两个噬菌体N4编码RNA聚合酶VRNAP和N4 RNAPII的激活。 3,500个氨基酸VRNAP识别其启动子的发夹和特定序列。启动子激活需要超螺旋和ECOSSB。我们定义并表征了一个活跃的中央结构域（1,106个小型VRNAP，是家族中最遥远的成员），并最近确定了其晶体结构，分辨率为2.0 A分辨率。 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相互作用引起了Ecossb辅助产品位移。 N4 RNAPII是一种异二聚体，无法识别启动子序列。在体内，它需要N4GP2，这是一种ssDNA结合蛋白，该蛋白专门募集N4 rnapii与ssDNA。中间启动子包含两组由12-25 bp隔开的保守序列。我们将确定所有N4 RNAPII启动子并分析其体内结构，以测试我们的启动子识别模型，确定N4 RNAPII的晶体结构以及与GP2的复合物的晶体结构，通过确定其本地MW来表征GP2，确定其本地MW，定义SSDNA结合的确定因素，并与RNAPII的相互作用以及与RNAPII的相互作用和GP2相互作用。我们将确定负责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，以重建具有纯化成分的系统。我们期望将新见解对启动子-RNAP相互作用的策略，依赖因子依赖性T7-1型RNA聚合酶的结构，以及DNA结构过渡和单链DNA结合蛋白在转录调节中的作用。