Novel Transcribing Activites in N4 Infected E. Coli

描述(申请人提供)：我们的工作集中在两个噬菌体N4编码的RNA聚合酶vRNAP和N4 RNAPII的结构、启动子识别和激活机制上，它们属于T7 RNAP样家族。3500个氨基酸的vRNAP识别发夹及其启动子的特定序列。启动子的激活需要超螺旋和EcoSSB。我们定义并表征了一个活性中心结构域(1,106个mini-vRNAP，该家族中距离最远的成员)，并于最近确定了其2.0A分辨率的晶体结构。我们将定义vRNAP启动子的体内结构以支持我们的超螺旋诱导发夹挤出模型，使用生化和遗传方法确定启动子识别的决定因素，通过X射线结晶学确定微型vRNAP-启动子DNA复合体的结构，定义用于延伸复合体结晶的核酸支架，并定义负责EcoSSB-vRNAP相互作用的氨基酸残基，从而导致EcoSSB辅助产物置换。N4 RNAPII是一种不识别启动子序列的异源二聚体。在体内，它需要N4gp2，一种ssDNA结合蛋白，特异性地将N4 RNAPII招募到ssDNA上。中间启动子含有两套相隔12-25个碱基对的保守序列。我们将鉴定所有N4 RNAPII启动子并分析它们的体内结构，以验证我们的启动子识别模型，确定N4 RNAPII及其与GP2的复合体的晶体结构，通过测定GP2的天然分子量来表征GP2，定义单链DNA结合和与RNAPII相互作用的决定因素，以及RNAPII中GP2相互作用的靶标。我们将鉴定负责N4 RNAPII启动子特异性的N4编码蛋白，并鉴定其与DNA、RNAPII和/或GP2的相互作用，以重建一个含有纯化成分的系统。我们期望为启动子-RNAP相互作用的策略，对依赖因子的T7-1ike RNA聚合酶的结构，以及DNA结构转变和单链DNA结合蛋白在转录调控中的作用提供新的见解。

DESCRIPTION (provided by applicant): Our work focuses on the structure, mechanism of promoter recognition and activation of two phage N4-coded RNA polymerases, vRNAP and N4 RNAPII, which belong to the T7 RNAP-like family. The 3,500 amino acid vRNAP recognizes a hairpin and specific sequences at its promoters. Promoter activation requires supercoiling and EcoSSB. We defined and characterized an active central domain (1,106 mini-vRNAP, the most distantly related member of the family) and have recently determined its crystal structure at 2.0 A resolution. We will define the in vivo structure of vRNAP promoters to support our model of supercoiled-induced hairpin extrusion, identify determinants of promoter recognition using biochemical and genetic approaches, determine the structure of the mini-vRNAP-promoter DNA complex by X-ray crystallography, define a nucleic acid scaffold for crystallization of the elongation complex, and define amino acid residues responsible for the EcoSSB-vRNAP interaction that elicits EcoSSB-assisted product displacement. N4 RNAPII is a heterodimer that does not recognize promoter sequences. In vivo it requires N4gp2, a ssDNA binding protein that recruits N4 RNAPII to ssDNA specifically. Middle promoters contain two sets of conserved sequences separated by 12-25 bp. We will identify all N4 RNAPII promoters and analyze their in vivo structure to test our model of promoter recognition, determine the crystal structure of N4 RNAPII and of its complex with gp2, characterize gp2 by determining its native MW, defining determinants of ssDNA-binding and of interaction with RNAPII, and the target of gp2 interaction in RNAPII. We will identify the N4-coded protein responsible for N4 RNAPII promoter specificity, and characterize its interaction with DNA, RNAPII and/or gp2 to reconstitute a system with purified components. We expect to provide new insights into strategies of promoter-RNAP interaction, into structure of factor-dependent T7-1ike RNA polymerases, and into the role of DNA structural transitions and single-stranded DNA binding proteins in transcription regulation.