NOVEL TRANSCRIBING ACTIVITIES IN N4 INFECTED E COLI

N4 感染大肠杆菌中的新转录活动

基本信息

批准号：
6322311
负责人：
LUCIA B. B ROTHMAN-DENES
金额：
$ 37.59万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-11-15 至 2003-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6322311
关键词：
DNA binding protein DNA directed RNA polymerase DNA footprinting Escherichia coli atomic force microscopy bacterial genetics coliphages crosslink enzyme activity fluorescence resonance energy transfer gene mutation genetic promoter element genetic transcription nucleic acid sequence protein structure function site directed mutagenesis virus genetics

项目摘要

Coliphage N4 transcription is carried out by three different RNA polymerases (RNAP) providing unique opportunities to study structure-function relationships in RNAPases, the role of DNA structure, supercoiling and single-stranded DNA binding (SSB) proteins in transcriptional activation. We propose to continue studies on the structure, mechanism of promoter recognition and activation of the virion-encapsidated, 320-kDa single-polypeptide RNAP (vRNAP) which is responsible for the synthesis of phage early RNAs. vRNAP recognizes a 5-7 bp stem, 3b loop hairpin and specific sequences at its promoters. N4 vRNAP promoters are activated by supercoiling and Eco SSB. Supercoiling leads to a Mg(II)- and sequence-dependent extrusion of the promoter hairpins. We have proposed that Eco SSB invades this structure with melting of the complementary strand hairpin and stabilization of the template strand hairpin which is recognized by vRNAP. We propose to characterize the DNA structural transition occurring at vRNAP promoters using fluorescence resonance transfer (FRET). We will further characterize the interaction vRNAP with its promoters by elucidating the sequence determinants of promoter binding through a binding site selection protocol and the use of a quantitative binding assay. The role of Eco SSB in providing the structure of the "activated promoter" will be defined by footprinting mapping of the topology of the "activated" promoter, determining the role of promoter downstream sequences and of single-strandedness in EcoSSB invasion and footprinting of vRNAP at the "activated promoter". The stoichiometry of Eco SSB binding at the activated promoter will be determined by scanning force microscopy. We will characterize the role of Eco SSB and promoter sequences in promoter clearance. Finally, we will define the vRNAP domain involved in promoter recognition through proteolysis and crosslinking studies. Results of these experiments will provide insights into a new strategy of promoter-RNAP interaction, novel determinants of protein-DNA interaction, as well as the role of single-stranded DNA binding proteins in regulation of gene expression.

Coliphage N4 转录由三种不同的 RNA 聚合酶 (RNAP) 进行，为研究 RNA 聚合酶的结构-功能关系、DNA 结构、超螺旋和单链 DNA 结合 (SSB) 蛋白在转录激活中的作用提供了独特的机会。我们建议继续研究负责噬菌体早期RNA合成的病毒体衣壳化的320kDa单多肽RNAP（vRNAP）的结构、启动子识别和激活机制。 vRNAP 可识别 5-7 bp 茎、3b 环发夹及其启动子处的特定序列。 N4 vRNAP 启动子由超螺旋和 Eco SSB 激活。超螺旋导致启动子发夹的 Mg(II) 和序列依赖性挤压。我们提出，Eco SSB 通过熔化互补链发夹并稳定 vRNAP 识别的模板链发夹来侵入该结构。我们建议使用荧光共振转移（FRET）来表征 vRNAP 启动子处发生的 DNA 结构转变。我们将通过结合位点选择方案和使用定量结合测定来阐明启动子结合的序列决定因素，从而进一步表征 vRNAP 与其启动子的相互作用。 Eco SSB 在提供“激活的启动子”结构中的作用将通过“激活的”启动子拓扑的足迹图谱来定义，确定启动子下游序列和单链在 EcoSSB 入侵和 vRNAP 足迹中的作用在“激活的启动子”处。 Eco SSB 在激活的启动子处结合的化学计量将通过扫描力显微镜测定。我们将描述 Eco SSB 和启动子序列在启动子清除中的作用。最后，我们将通过蛋白水解和交联研究来定义参与启动子识别的 vRNAP 结构域。这些实验的结果将为了解启动子-RNAP相互作用的新策略、蛋白质-DNA相互作用的新决定因素以及单链DNA结合蛋白在基因表达调节中的作用提供见解。