NOVEL TRANSCRIBING ACTIVITIES IN N4 INFECTED E COLI

大肠杆菌噬菌体N4的转录是由三种不同的RNA聚合酶（RNAP）进行的，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（SSB）蛋白在转录激活中的作用提供了独特的机会。 我们建议继续研究的结构，启动子识别和激活的病毒粒子的，320 kDa的单多肽RNAP（vRNAP），这是负责噬菌体早期RNA的合成机制。 vRNAP识别5-7 bp茎、3b环发夹和其启动子处的特异性序列。 N4 vRNAP启动子通过超螺旋和Eco SSB激活。 超螺旋导致启动子发夹的Mg（II）和序列依赖性挤出。 我们已经提出，Eco SSB通过互补链发夹的解链和被vRNAP识别的模板链发夹的稳定化侵入该结构。我们建议使用荧光共振转移（FRET）来表征在vRNAP启动子处发生的DNA结构转变。 我们将通过阐明启动子结合的序列决定因素，通过结合位点选择方案和使用定量结合试验，进一步表征vRNAP与其启动子的相互作用。 Eco SSB在提供“活化的启动子”的结构中的作用将通过“活化的”启动子的拓扑结构的足迹作图来定义，确定启动子下游序列和单链在EcoSSB侵入中的作用以及vRNAP在“活化的启动子”处的足迹。 将通过扫描力显微镜测定在活化的启动子处结合的Eco SSB的化学计量。 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。这些实验的结果将提供一个新的策略的启动子RNAP相互作用，蛋白质-DNA相互作用的新的决定因素，以及单链DNA结合蛋白在基因表达调控的作用的见解。

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.