NOVEL TRANSCRIBING ACTIVITIES IN N4-INFECTED E COLI

我们将使用生物化学，遗传学和物理方法来分析 结构、功能、启动子利用机制及调控 需要两种噬菌体编码的DNA依赖性RNA聚合酶的活性 在噬菌体N4的发育过程中。 早期N4 RNA由病毒体包裹的噬菌体编码的320- kDa的单多肽RNA聚合酶（vRNAP），它既识别 结构和启动子上的特异性序列 模板，即，它是一种位点特异性单链DNA结合 蛋白 双链DNA上的启动子识别需要 超螺旋模板和E.大肠杆菌单链DNA结合蛋白 （SSB）。 激活是E. coli SSB. DNA结构在 启动子模板链将通过SELEX、凝胶迁移率和 核磁共振波谱法。 超螺旋和SSB在N4激活中的作用 vRNAP启动子将通过化学反应性、足迹法 和动力学分析。 我们将描述SSB突变体， 特异性影响N4早期RNA合成，以了解为什么SSB而不是 其他单链DNA结合蛋白可以激活N4 vRNAP 发起人。 将完成vRNAP基因的测序，以便 通过基因操作来确定蛋白质的结构域 负责启动子识别。 最后，我们会展开研究， 探讨N4 vRNAP终止转录的机制。 中间N4 RNA由第二个噬菌体编码的RNA聚合酶合成 (N4 RNAP II）由30-和40-kDa的两种多肽组成。 第三个15 kDa 蛋白质允许N4 RNAPII识别它的启动子， 在彼此相距可变距离的两个嵌段处的同源性 （t/aAAAT在+1和Tt/aCTGGACa/t，上游14-21 bp）。 我们将确定 保守序列与启动子活性的相关性（和 它们之间的距离）在转录起始位点， 缺失和突变分析。 15-kDa的相互作用 具有启动子的多肽的特征在于凝胶阻滞， 足迹法和特异性突变体的分离。 我们将 启动对15-kDa多肽的结构研究。 我们最近 显示，虽然异二聚体，N4 RNAP II共享序列同源性 与T7样和酵母线粒体RNA聚合酶，因此， 是这个家庭中最小的成员 我们将启动结构和 N4 RNAP的功能研究II. 我们将确定它与 通过足迹法和交联法将15-kDa多肽定位在启动子上 分析和定位的初始核苷酸结合位点，DNA 结合位点和RNA结合。

We will use biochemical, genetic and physical approaches to analyze the structure, function, mechanism of promoter utilization and regulation of activity of two phage-coded, DNA-dependent RNA polymerases required during bacteriophage N4 development. Early N4 RNAs are transcribed by a virion-encapsulated, phage-coded, 320- kDa single-polypeptide RNA polymerase (vRNAP) which recognizes both a structure and specific sequences at its promoters on single-stranded template, i.e., it is a site-specific, single-stranded DNA binding protein. Promoter recognition on double-stranded DNA requires supercoiled template and E. coli single-stranded DNA binding protein (SSB). Activation is specific for E. coli SSB. The DNA structure at the promoter template strand will be determined by SELEX, gel mobility and NMR spectroscopy. The role of supercoiling and SSB in activation of N4 vRNAP promoter swill be determined by chemical reactivity, footprinting and kinetic analysis. We will characterize SSB mutants which specifically affect N4 early RNA synthesis to understand why SSB but not other single-stranded DNA binding proteins can activate N4 vRNAP promoters. The sequence of the vRNAP gene will be completed in order to manipulate it genetically to determine the domain of the protein responsible for promoter recognition. Finally, we will initiate studies to investigate the mechanism of transcription termination by N4 vRNAP. Middle N4 RNAs are synthesized by a second phage-coded, RNA polymerase (N4 RNAP II) composed of two polypeptides 30- and 40-kDa. A third 15-kDa protein allows N4 RNAPII to recognize its promoters which share sequence homology at two blocks present at variable distance from each other (t/aAAAT at +1 and Tt/aCTGGACa/t, 14-21 bp upstream). We will determine the relevance to promoter activity of the conserved sequences (and distance between them) at the sites of transcription initiation by deletion and mutation analysis. The interaction of the 15-kDa polypeptide with promoters will be characterized by gel retardation, footprinting and the isolation of change of specificity mutants. We will initiate structural studies on the 15-kDa polypeptide. We have recently shown that, although heterodimeric, N4 RNAP II shares sequence homology with the T7-like and yeast mitochondrial RNA polymerases and therefore, is the smallest member of this family. We will initiate structural and functional studies on N4 RNAP II. We will determine its interaction with the 15-kDa polypeptide at the promoter by footprinting and crosslinking analysis and the localization of the initial nucleotide binding site, DNA binding site and RNA binding using photocrosslinking nucleotide analogs.