NOVEL TRANSCRIBING ACTIVITIES IN N4 INFECTED E COLI

噬菌体N4的转录由三种不同的RNA聚合酶(RNAP)进行，这为研究RNAP酶的结构-功能关系、DNA结构、超螺旋和单链DNA结合蛋白(SSB)在转录激活中的作用提供了独特的机会。我们建议继续研究病毒粒子包裹的320 kDa单肽RNAP(VRNAP)的结构、启动子识别和激活机制，vRNAP负责合成噬菌体早期RNA。VRNAP识别5-7bp的茎、3b环发夹及其启动子上的特定序列。N4 vRNAP启动子由超级卷曲和Eco SSB激活。超螺旋导致启动子发夹的依赖于镁(II)和序列的挤出。我们提出了Eco SSB通过融化互补链发夹和稳定模板链发夹来入侵这一结构，这是vRNAP所识别的。我们建议用荧光共振转移(FRET)来表征发生在vRNAP启动子上的DNA结构转变。我们将通过结合位点选择协议和定量结合分析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“激活启动子”结构中的作用将通过绘制“激活”启动子的拓扑图、确定启动子下游序列和单链在EcoSSB入侵中的作用以及在“激活启动子”处vRNAP的足迹来确定。Eco SSB结合在激活启动子上的化学计量比将通过扫描力显微镜来确定。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，我们将通过蛋白分解和交联研究来确定参与启动子识别的vRNAP结构域。这些实验的结果将为启动子-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控中的作用提供见解。

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.