GENE REGULATION BY TRANSCRIPTION ANTITERMINATION

这项提议的长期目标是获得一个完整的 对细菌转录终止调控的认识 原核细胞。这个实验室一直致力于阐明 某些转录激活蛋白的作用机制 抑制转录机器的识别能力 并对终止和释放的遗传信号作出反应 MRNA.本研究所使用的模型系统为噬菌体lambda 其基因N产物被假设为作用于RNA 位于特定顺式作用位点的聚合酶，以允许形成 抗终止转录装置。修改后的 N蛋白对转录装置的影响涉及许多 其中一些起到积极作用的细胞蛋白质 末端和其他作为核糖体的组成部分。 手机。拟议的实验，结合了遗传、生化 和免疫化学方法，将试图识别所有 参与N依赖抗终止的细胞成分 过程，确定这些因素是否以及哪些是 正常转录和N-修饰延伸转录的组成 设备，并确定它们的组装顺序和 允许它们组装的各个识别信号。 相互作用蛋白的结构域及其各自的基因 信号将通过对抑制器的分析来识别 突变，基因融合，一种专门设计的结构 转录因子的嵌合体，并通过使用 化学合成的多肽和寡核苷酸。这些网站 在各自蛋白质的相互作用区域中的接触 遗传信号将由化学物质和紫外线决定 交叉链接研究，并通过故意改变这些 实体通过定点突变。假设 N蛋白，作为伸长转录的稳定亚基 设备，与终止信号和因素竞争 与RNA聚合酶终止域的相互作用将 接受测试。除了提供一种分子机制， 终止抑制，这些研究应该提供基本的 关于加长的化学性质的信息 转录装置和生理参数以及 控制转录延长的遗传信号 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.