GENE REGULATION BY TRANSCRIPTION ANTITERMINATION

该提案的长期目标是获得一个完整的 理解转录终止的调控， 原核细胞 该实验室致力于阐明 某些转录激活蛋白的作用机制 抑制转录机制识别 并对基因信号作出反应， mRNA。 本研究中使用的模型系统是λ噬菌体 其基因N产物被假设作用于RNA 聚合酶在特定的顺式作用位点，以允许形成一个 终止抗性转录器。 修改 N蛋白对转录器的影响涉及许多 细胞蛋白质，其中一些起积极作用， 终止和其他作为核糖体的组成部分， cell. 所提出的实验，结合遗传，生化 和免疫化学方法，将试图确定所有 参与N依赖性抗终止的细胞成分 过程，确定这些因素是否以及哪些因素是 正常和N修饰的延伸转录的组分 他们的目的是为了确定他们的目的，并确定他们的目的。 允许它们组装的相应识别信号。 相互作用蛋白质的结构域和各自的遗传结构域是相互作用的蛋白质的结构域。 信号将通过分析抑制器来识别 突变，基因融合，构建专门设计的 转录因子的嵌合体，并通过使用 化学合成的肽和寡核苷酸。 网站 在各自蛋白质的相互作用结构域中的接触 基因信号将由化学和紫外线决定 交联研究，并通过故意改变这些 实体通过定点诱变。 的假设 N蛋白作为延伸转录的稳定亚基 设备，与终止信号和因素竞争， 与RNA聚合酶的终止结构域的相互作用将 得到考验 除了提供一个分子机制， 终止抑制，这些研究应该提供基本的 关于延伸的化学性质的信息 转录器和生理参数， 控制转录延伸的遗传信号 原核细胞

The long term goal of this proposal is to obtain a complete understanding of the regulation of transcription termination in a procaryotic cell. This laboratory has focused on elucidating the mechanism by which certain transcriptional activator proteins act to suppress the ability of the transcription machinery to recognize and respond to genetic signals for termination and release of mRNA. The model system used in this study is phage lambda whose gene N product has been hypothesized to act on RNA polymerase at specific cis-acting loci to allow the formation of a termination-resistant transcription apparatus. The modification of the transcription apparatus by N protein involves a number of cellular proteins of which some act to positively effect termination and others act as components of the ribosome in the cell. The proposed experiments, combining genetic, biochemical and immunochemical approaches, will attempt to identify all cellular components involved in N-dependent antitermination process, determine whether and which of these factors are components of the normal and N-modified elogating transcription apparatus, and determine the order with which they assemble and the respective recognition signals which allow their assembly. The domains of the interacting proteins and respective genetic signals will be identified through the analysis of suppressor mutations, gene fusions an construction of specifically designed chimeras of the transcription factors, and by employing chemically synthesized peptides and oligonucleotides. The sites of contacts in the interacting domains of the respective proteins and the genetic signals will be determined by chemical and UV cross-linking studies, and by deliberate alterations of these entities through site-directed mutagenesis. The hypothesis that the N protein, as a stable subunit of the elongating transcription apparatus, competes with termination signals and factors for interaction with the termination domain of RNA polymerase will be tested. In addition to providing a molecular mechanism of termination suppression, these studies should provide fundamental information on the chemical nature of the elongating transcription apparatus and the physiological parameters and the genetic signals which govern transcription elongation in the procaryotic cell.