STRUCTURE/FUNCTION OF TRANSCRIPTION COMPLEX RNA HAIRPINS

这个项目的目标是定义 幼体中二级结构(RNA发夹)的形成 转录会导致转录过程中的暂停和终止。 RNA二级结构是细胞内重要的调控信号 转录暂停和终止的原核生物 基因调控机制的主要组成部分；暂停和 在真核生物中也会发生过早终止的情况，在那里他们的 监管的重要性现在才开始显现。建议数 研究将提供对RNA发夹如何 形成以及它们如何在转录复合体中相互作用。 理解这个模型系统将是一个重要的 对转录调控知识的贡献， 控制所有基因表达的基本过程。这些研究还包括 将改进技术和概念，用于 真核转录复合体的未来研究。 生化、遗传和重组DNA的组合 技术将被用于研究rna发夹转录。 复杂的互动。模型RNA的系统变异 发夹将定义序列和结构要求 RNA发夹诱导转录暂停和终止 队形。一个主要目标将是确定是否形成了 RNA发夹只能通过改变导致暂停和终止 转录复合体或IF中的核酸构象 转录过程中蛋白质间的特异性相互作用 涉及复合体和部分RNA发夹。核糖核酸 发夹指定区域将由合成的 将寡核苷酸插入到特殊的载体前面 T7 A1启动子的启动，以便于研究它们的暂停和 终止表型。转录复合体，包含 已定义的新生转录RNA发夹将从 体外转录反应，并经历广泛的结构 用酶和化学探针分析RNA和DNA 结构。将对RNA聚合酶的β亚基进行分析 通过对其基因进行定点突变来确定 氨基酸残基可能参与RNA发夹相互作用。

The goal of this project is to define the mechanisms by which formation of secondary structures (RNA hairpins) in nascent transcripts causes pausing and termination during transcription. RNA secondary structures are important regulatory signals in prokaryotes where transcription pausing and termination are major components of genetic regulatory mechanisms; pausing and premature termination also occur in eukaryotes where their regulatory significance is only now emerging. The proposed studies will provide a detailed understanding of how RNA hairpins form and how they interact in the transcription complex. Understanding this model systems will be an important contribution to knowledge about transcription regulation, the basic process that governs all gene expression. These studies also will refine techniques and concepts that will be employed in future work on eukaryotic transcription complexes. A combination of biochemical, genetic, and recombinant DNA techniques will be used to study RNA hairpin-transcription complex interactions. Systematic variation of model RNA hairpins will define the sequence and structural requirements for induction of transcription pausing and termination by RNA hairpin formation. A major goal will be to determine if the formation of RNA hairpins causes pausing and termination only by altering nucleic acid conformations in the transcription complex or if specific interactions between proteins in the transcription complex and portions of the RNA hairpin are involved. RNA hairpin-specifying regions will be constructed from synthetic oligonucleotides and inserted into special plasmid vectors in front of the T7 A1 promoter to facilitate study of their pausing and termination phenotypes. Transcription complexes containing defined nascent transcript RNA hairpins will be isolated from in vitro transcription reactions and subjected to extensive structural analyses using enzymatic and chemical probes of RNA and DNA structure. The beta subunit of RNA polymerase will be analyzed by site-directed mutagenesis of its gene to determine which amino acid residues may participate in RNA hairpin interactions.