Novel Transcribing Activites in N4 Infected E. Coli

描述（由申请人提供）：我们的工作集中于两种噬菌体N4编码的RNA聚合酶的结构、启动子识别和激活机制，vRNAP和N4 RNAPII，它们属于T7 RNAP样家族。3,500个氨基酸的vRNAP识别发夹和其启动子处的特定序列。启动子激活需要超螺旋和EcoSSB。我们定义并表征了一个活性中心结构域（1，106个mini-vRNAP，家族中最远的成员），最近在2.0 A分辨率下确定了其晶体结构。我们将定义vRNAP启动子的体内结构以支持我们的超卷曲诱导的发夹挤出模型，使用生物化学和遗传学方法鉴定启动子识别的决定因素，通过X射线晶体学确定mini-vRNAP-启动子DNA复合物的结构，定义用于延伸复合物结晶的核酸支架，并定义负责EcoSSB-vRNAP相互作用的氨基酸残基，所述相互作用促进EcoSSB-assisted产物置换。N4 RNAPII是不识别启动子序列的异二聚体。在体内，它需要N4 gp 2，一种特异性地将N4 RNAPII募集到ssDNA的ssDNA结合蛋白。中间启动子含有两组相隔12-25 bp的保守序列。我们将确定所有N4 RNAPII启动子和分析其体内结构，以测试我们的启动子识别模型，确定N4 RNAPII及其与gp 2复合物的晶体结构，通过确定其天然分子量来表征gp 2，定义ssDNA结合和与RNAPII相互作用的决定因素，以及RNAPII中gp 2相互作用的靶点。我们将确定负责N4 RNAPII启动子特异性的N4编码蛋白，并表征其与DNA，RNAPII和/或gp 2的相互作用，以重建具有纯化组分的系统。我们希望提供新的见解启动子-RNAP相互作用的策略，到因子依赖的T7- 1样RNA聚合酶的结构，并到DNA结构转换和单链DNA结合蛋白在转录调控中的作用。

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.