Novel Transcribing Activites in N4 Infected E. Coli

描述（由申请人提供）：我们的工作重点是研究两种噬菌体N4编码RNA聚合酶vRNAP和N4 RNAPII的结构、启动子识别和激活机制，这两种酶属于T7 rnap样家族。由3500个氨基酸组成的vRNAP识别发夹和启动子上的特定序列。启动子激活需要超卷绕和EcoSSB。我们定义并表征了一个活跃的中心域（1106个迷你vrnap，家族中最远亲的成员），最近以2.0 A分辨率确定了它的晶体结构。我们将定义vRNAP启动子的体内结构，以支持我们的超线圈诱导发夹挤压模型，使用生化和遗传学方法确定启动子识别的决定因素，通过x射线晶体学确定迷你vRNAP-启动子DNA复合物的结构，定义核酸支架的结晶延伸复合物。并定义了导致ecossb辅助产物置换的EcoSSB-vRNAP相互作用的氨基酸残基。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，可以特异性地将N4 RNAPII招募到ssDNA上。中间启动子包含两组相隔12 ~ 25bp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来测试我们的启动子识别模型，确定N4 RNAPII及其与gp2复合物的晶体结构，通过测定其天然分子量来表征gp2，确定ssdna结合和与RNAPII相互作用的决定因素，以及RNAPII中gp2相互作用的目标。我们将鉴定负责N4 RNAPII启动子特异性的N4编码蛋白，并表征其与DNA， RNAPII和/或gp2的相互作用，以重建纯化组分的系统。我们期望在启动子- rnap相互作用策略、因子依赖性t7 -1样RNA聚合酶结构、DNA结构转变和单链DNA结合蛋白在转录调控中的作用等方面提供新的见解。

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.