ACTIVATION OF RNA POLYMERASE BY SSDNA BINDING PROTEIN

描述：噬菌体N4单链DNA结合蛋白 (N4SSB)对于噬菌体复制、重组和， 出乎意料的是，抄写晚了。我们将使用生化遗传和 分析N4SSB结构及其机理的物理方法 被识别的噬菌体N4晚期启动子的激活 通过大肠杆菌Sigma-70RNA聚合酶(RNAP)。N4SSB不是一个站点- 特异性dsDNA结合蛋白。我们已经分离出N4SSB羧基末端 突变体，它们擅长复制和重组，但 在体内和体内支持晚期转录激活的缺陷 体外培养。相反，缺乏单链DNA结合的突变体不支持 复制和重组，但激活体内转录和 体外实验表明转录的单链DNA结合活性 激活。最近的生化结果表明，N4SSB激活了 通过与E.ColiSigma-70RNAP直接相互作用转录。 N4SSB激活缺陷突变体不与RNAP结合。我们建议： 1)鉴定N4SSB与大肠杆菌RNAP相互作用的靶标 生物化学和遗传方法。为此，我们将确定 N4SSB亚基在大肠杆菌RNAP中的亲和作用 层析和交联，以及N4SSB之间的结合常数 和大肠杆菌RNAP的荧光发射各向异性。我们将隔离 对N4SSB激活不敏感的新RNAP突变体和E.coliRNA N4SSB激活缺陷突变体的聚合酶抑制物。我们会 397C RNAP的特性鉴定及新的C末端RNAP突变体的分离 贝塔‘亚单位。2)鉴定N4SSB的激活结构域。 我们将从生物化学的角度定义N4SSB的结构域，克隆和过度生产 N4SSB的结构域，并测定其体内和体外效应 转录上的结构域。3)确定所需的启动子序列 通过研究N4SSB对E. ColiRNAP在一组特性良好的启动子和鉴定 N4SSB必需的N4晚期启动子序列 激活。4)确定N4SSB对启动子结合的影响，开放 用复合体形成和启动子清除来鉴定RNAP中的STEP 它是N4SSB的作用部位。此外， 将研究超级卷曲对这些步骤的贡献。5) 鉴定大肠杆菌RNAP相关因子在N4晚期的作用 抄写。6)启动N4SSB的结构研究。我们期待着 这些拟议的研究将为我们提供对 原核生物和真核生物中的转录激活。

DESCRIPTION: The bacteriophage N4 single-stranded DNA binding protein (N4SSB) is essential for phage replication, recombination and, unexpectedly, late transcription. We will use biochemical genetic and physical approaches to analyze the structure of N4SSB and its mechanism of activation at the bacteriophage N4 late promoters which are recognized by the E. coli sigma-70 RNA polymerase (RNAP). N4SSB is not a site- specific dsDNA binding protein. We have isolated N4SSB carboxy-terminal mutants, which are proficient for replication and recombination but deficient in supporting late transcription activation in vivo and in vitro. In contrast, mutants deficient in ssDNA binding do not support replication and recombination but activate transcription in vivo and invitro indicating that the ssDNA binding activity of transcription activations. Recent biochemical results indicate that N4SSB activates transcription by interacting directly with E. coli sigma-70 RNAP. N4SSB activation-deficient mutants do not bind to RNAP. We propose to: 1) Identify the target of N4SSB interaction with E. coli RNAP by biochemical and genetic approaches. To that end we will determine the subunit which is the target of N4SSB in E. coli RNAP by affinity chromatography and crosslinking, and the binding constant between N4SSB and E. coli RNAP by fluorescence emmission anisotropy. We will isolate new RNAP mutants insensitive to N4SSB activation, and E. coli RNA polymerase suppressors of N4SSB activation- deficient mutants. We will characterize 397C RNAP and isolate new RNAP mutants in the C-terminus of the beta' subunit. 2) Characterize the activation domain of N4SSB. We will define biochemically the domain of N4SSB, clone and overproduced the domains, and determine the in vivo and in vitro effects of the N4SSB domains on transcription. 3) Determine promoter sequences required for N4SSB activation by studying the effect of N4SSB on the activity of E. coli RNAP at a set of well characterized promoters and identifying sequences at the N4 late promoters which are essential for N4SSB activation. 4) Determine the effect of N4SSB on promoter binding, open complex formation and promoter clearance to identify the steop in RNAP initiation which is the site of action of N4SSB. In addition, the contribution of supercoiling to these steps will be studied. 5) Characterize the role of E. coli RNAP associated factors in N4 late transcription. 6) Initiate structural studies of N4SSB. We expect that the proposed studies will provide new insights into the mechanism of transcription activation in prokaryotes as well as eukaryotes.