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，后期转录。 我们将使用生化遗传学和 分析N4SSB结构和机理的物理方法 噬菌体N4晚期启动子的激活， 由大肠coli sigma-70 RNA聚合酶（RNAP）。 N4SSB不是一个网站- 特异性dsDNA结合蛋白。 我们分离出了N4SSB羧基端 突变体，它们精通复制和重组， 缺乏支持体内晚期转录激活， 体外 相反，ssDNA结合缺陷的突变体不支持 复制和重组，但在体内激活转录， 体外表明转录的ssDNA结合活性 激活。 最近的生化结果表明，N4SSB激活 通过直接与E. coli sigma-70 RNAP。 N4SSB激活缺陷型突变体不与RNAP结合。 我们建议： 1)确定N4SSB与E.大肠杆菌RNAP， 生物化学和遗传学方法。 为此，我们将确定 N4SSB的作用靶点是E.大肠杆菌RNAP亲和法 色谱和交联，和N4SSB之间的结合常数 和E. coli RNAP的荧光发射各向异性。 我们将隔离 新的RNAP突变体对N4SSB激活不敏感，E.杆菌RNA N4SSB活化缺陷突变体的聚合酶抑制子。 我们将 表征397 C RNAP并分离C-末端的新RNAP突变体 β亚基的一部分 2)表征N4SSB的激活结构域。 我们将用生物化学方法定义N4SSB的结构域，克隆和过量生产 结构域，并确定N4SSB的体内和体外作用 域的转录。 3)确定启动子序列所需 通过研究N4SSB对E. coli RNAP在一组充分表征的启动子上的表达，并鉴定 N4晚期启动子上的序列，其对于N4SSB是必需的 activation. 4)确定N4SSB对启动子结合、开放 复合物的形成和启动子清除，以确定RNAP中的steps N4SSB的起始位点。 此外该 将研究超螺旋对这些步骤的贡献。 第五章） 描述了E. N4晚期大肠杆菌RNAP相关因子 转录。 6)启动N4SSB的结构研究。 我们预计 拟议的研究将提供新的见解， 在原核生物以及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.