ACTIVATION OF RNA POLYMERASE BY SSDNA BINDING PROTEIN

SSDNA 结合蛋白激活 RNA 聚合酶

• 批准号: 2701762
• 负责人: LUCIA B. B ROTHMAN-DENES
• 金额: $ 24.07万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2701762
• 关键词: DNA binding protein; DNA directed RNA polymerase; Escherichia coli; affinity chromatography; bacterial proteins; crosslink; enzyme activity; genetic promoter element; genetic recombination; genetic transcription; nucleic acid sequence; nucleic acid structure; protein structure function; virus replication

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.

描述：噬菌体 N4 单链 DNA 结合蛋白 (N4SSB) 对于噬菌体复制、重组至关重要， 没想到，转录晚了。 我们将利用生化遗传和 物理方法分析N4SSB的结构及其机理 被识别的噬菌体 N4 晚期启动子的激活 由大肠杆菌 sigma-70 RNA 聚合酶 (RNAP) 产生。 N4SSB 不是一个站点- 特异性双链DNA结合蛋白。 我们分离出 N4SSB 羧基末端 突变体，擅长复制和重组，但 缺乏支持体内和体内的晚期转录激活 体外。 相反，单链DNA结合缺陷的突变体不支持 复制和重组，但激活体内转录 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 通过直接与大肠杆菌 sigma-70 RNAP 相互作用进行转录。 N4SSB 激活缺陷突变体不与 RNAP 结合。 我们建议： 1) 确定 N4SSB 与大肠杆菌 RNAP 相互作用的靶标 生物化学和遗传学方法。 为此，我们将确定 通过亲和力，它是大肠杆菌 RNAP 中 N4SSB 的靶标亚基 色谱和交联，以及 N4SSB 之间的结合常数 和大肠杆菌 RNAP 通过荧光发射各向异性。 我们将隔离 对 N4SSB 激活不敏感的新 RNAP 突变体和大肠杆菌 RNA N4SSB 激活缺陷突变体的聚合酶抑制剂。 我们将 表征 397C RNAP 并在 C 末端分离新的 RNAP 突变体 β' 亚基。 2）表征N4SSB的激活域。 我们将从生化角度定义 N4SSB、克隆和过量生产的结构域 域，并确定 N4SSB 的体内和体外效应 转录域。 3) 确定所需的启动子序列 通过研究 N4SSB 对大肠杆菌活性的影响来激活 N4SSB。 大肠杆菌 RNAP 在一组特征明确的启动子上并鉴定 N4 晚期启动子上的序列对于 N4SSB 至关重要 激活。 4）确定N4SSB对启动子结合的影响，打开 复合物形成和启动子清除以识别 RNAP 中的步骤 起始点是 N4SSB 的作用位点。 此外， 将研究超螺旋对这些步骤的贡献。 5） 表征大肠杆菌 RNAP 相关因子在 N4 晚期的作用 转录。 6) 启动N4SSB的结构研究。 我们期望 拟议的研究将为该机制提供新的见解 原核生物和真核生物中的转录激活。