STRUCTURE/FUNCTION OF TRANSCRIPTION COMPLEX RNA HAIRPINS

该项目的目标是确定机制， 新生细胞中二级结构（RNA发夹）的形成 转录物在转录期间引起暂停和终止。 RNA的二级结构是重要的调控信号， 在原核生物中， 基因调控机制的主要组成部分;暂停和 真核生物中也会发生早产， 监管意义现在才显现出来。 拟议 研究将提供一个详细的了解如何RNA发夹 以及它们在转录复合体中如何相互作用。 了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 将完善技术和概念，将用于 真核生物转录复合体的未来研究。 生物化学、遗传学和重组DNA的组合 技术将用于研究RNA发夹转录 复杂的互动。 模式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 阵 一个主要的目标将是确定是否形成 RNA发夹结构仅通过改变 转录复合物中的核酸构象，或者如果 转录过程中蛋白质之间的特异性相互作用 复合物和RNA发夹的部分参与。 RNA 发夹特异性区域将由合成的 寡核苷酸并插入到前面的特殊质粒载体中 的T7 A1启动子，以促进其暂停和 终止表型 转录复合物， 定义的新生转录RNA发夹将从 体外转录反应和广泛的结构 使用RNA和DNA的酶和化学探针进行分析 结构 将分析RNA聚合酶的β亚基 通过其基因的定点突变来确定 氨基酸残基可以参与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.