STRUCTURE/FUNCTION OF TRANSCRIPTION COMPLEX RNA HAIRPINS

这个项目的长期目标是定义 通过RNA聚合酶引起暂停和终止的转录复合物。 RNA二级结构是原核生物中重要的调控信号， 其中转录暂停和终止是 基因调控机制。 暂停和提前终止也 影响哺乳动物细胞和病毒的基因表达。 暂停的变化 或终止影响基因的表达， 癌症的发展和艾滋病病毒HIV-1的生长。 的 哺乳动物细胞中暂停和终止的基本机制是 未知和难以研究。 专业的系统更容易 调查 该项目将进一步了解转录 在E.大肠杆菌，这是一个很好的模型系统， 研究转录复合体中的基本相互作用。 工作 还将提供技术和概念， 促进真核转录复合体的工作。 将采用生物化学和遗传学相结合的方法， 细菌转录复合体中的相互作用。 系统变化 的暂停和终止RNA发夹序列和结构将定义 影响RNA聚合酶延伸的特征。 定义 将分离转录复合物，并使用 一系列可以揭示RNA和DNA结构的试剂 其中的一根。 这些生物化学研究得到了补充， RNA聚合酶的遗传分析。 β和β 1亚基 包含氨基酸变化改变停顿识别的区域， 终止信号。 一组突变的酶， 不同的地区将被隔离和研究，以揭示潜在的作用， 在暂停和终止点发生的事件中的每一个。 这些 实验将用于测试转录的替代模型， 终止，一个简单碱基配对平衡解释 第二，终止是多步骤过程， RNA聚合酶活跃地催化。

The long-range goal of this project is to define the interactions in the transcription complex that cause pausing and termination by RNA polymerase. RNA secondary structures are important regulatory signals in prokaryotes, where transcriptional pausing and termination are major components of genetic regulatory mechanisms. Pausing and premature termination also affect gene expression in mammalian cells and viruses. Changes in pausing or termination influence expression of genes that are involved in development of cancer and in the growth of the AIDS virus, HIV-1. the underlying mechanisms for pausing and termination in mammalian cells are unknown and difficult to study. Prokaryotic systems are more amendable to investigation. This project will further understanding of transcriptional pausing and termination in E. coli, which is an excellent model system for studying the fundamental interactions in the transcription complex. Work on the model system also will provide techniques and concepts that will facilitate work on eukaryotic transcription complexes. A combined biochemical and genetic approach will be used to dissect the interactions in the bacterial transcription complex. Systematic variation of pause and termination RNA hairpin sequences and structures will define the features that influence elongation by RNA polymerase. Defined transcription complexes will be isolated and their structures probed using a variety of agents that can reveal the configuration of RNA and DNA strands within them. These biochemical studies are complemented by a genetic analysis of RNA polymerase. Both the beta and beta1 subunits contain regions where amino-acid changes alter recognition of the pause and termination signals. A set of mutant enzymes with changes in these different regions will be isolated and studied to reveal the potential role of each in the events that occur at the pause and termination sites. These experiments will be used to test alternative models for transcriptional termination, one that simple base-pairing equilibria account for termination and the second that termination is a multi-step process that RNA polymerase actively catalyzes.