NOVEL TRANSCRIBING ACTIVITIES IN N4 INFECTED E COLI

噬菌体N4转录由三种不同的RNA聚合酶（RNAP）进行，为研究rnapase的结构-功能关系、DNA结构、超卷曲和单链DNA结合（SSB）蛋白在转录激活中的作用提供了独特的机会。我们建议继续研究病毒包膜的320 kda单多肽RNAP （vRNAP）的结构、启动子识别和激活机制，vRNAP负责噬菌体早期rna的合成。vRNAP识别5- 7bp的茎，3b的环状发夹和启动子上的特定序列。N4 vRNAP启动子由超卷曲和Eco SSB激活。超卷曲导致启动子发夹的Mg(II)-和序列依赖挤出。我们提出Eco SSB通过vRNAP识别的互补链发卡的熔化和模板链发卡的稳定侵入该结构。我们建议使用荧光共振转移（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方案和定量结合试验阐明启动子结合的序列决定因素，进一步表征vRNAP与其启动子的相互作用。EcoSSB在提供“激活启动子”结构中的作用将通过“激活”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性在EcoSSB入侵和vRNAP在“激活启动子”的足迹图谱中的作用。Eco SSB在激活启动子上的结合化学计量学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.