RECOGNITION AND CATALYSIS IN RNASE III PROCESSING

长期目标是确定RNA序列和结构 核糖核酸酶III加工底物中的元件，其指导特异性 结合酶并促进加工催化。 核糖核酸酶III 底物是噬菌体T7 R1.1处理信号， T7早期mRNA前体中含有的60个核苷酸的RNA发夹。 的 R1.I底物在RNA酶III的单个特异性位点上进行切割， 体外，基因调控的后果。 具体目标是： . 鉴定直接参与结合RNase III的核苷酸， 通过RNase III-R1. 1 RNA复合物的化学和酶探测。 . 识别形成RNase的RNA碱基对和功能基团 第三，识别要素，通过对加工反应性的分析， R1.1序列变体的结合亲和力。 . 确定镁离子参与底物识别， 使用RNase III点突变体（Rnc70），其结合底物，但 不能被Mg 2+催化裂解。 正在进行的R1.1处理信号的合作质子NMR分析， 核糖核酸酶Ⅲ及其与底物复合物的X射线晶体学研究 将提供重要的结构信息，以补充生物化学 问题研究 该项目对以下方面具有额外的重要影响： 人类健康和疾病。 具体来说，RNase III已被证明具有 与其他dsRNA结合的强结构和/或功能相似性 蛋白质，包括一种哺乳动物的核蛋白， 免疫缺陷病毒（HIV）TAR元件，和哺乳动物dsRNase， 作为抗病毒反应的一部分。 理解 RNase III的加工催化作用可以提供一个定义的基础， 蛋白质-dsRNA相互作用在人类健康和疾病中的作用。

The long-term objective is to determine the RNA sequence and structural elements in a ribonuclease III processing substrate that direct specific binding of enzyme and promote processing catalysis. The ribonuclease III substrate is the bacteriophage T7 R1.1 processing signal, an approximately 60 nucleotide RNA hairpin contained in the T7 early mRNA precursor. The R1.I substrate undergoes cleavage by RNase III at a single specific site in vitro, with gene regulatory consequences. The specific aims are to: . Identify the nucleotides that directly participate in binding RNase III, through chemical and enzymatic probing of the RNase III-R1.1 RNA complex. . Identify the RNA base-pairs and functional groups that form the RNase III recognition elements, through analyzing the processing reactivities and binding affinities of R1.1 sequence variants. . Determine the involvement of magnesium ion in substrate recognition by employing an RNase III point mutant (Rnc70) which binds substrate, but is incapable of Mg2+-catalyzed cleavage. Ongoing collaborative proton NMR analysis of the R1.1 processing signal and an x-ray crystallographic study of RNase III and its complex with substrate will provide important structural information to complement the biochemical studies. This project has additional important implications regarding human health and disease. Specifically, RNase III has been shown to have strong structural and/or functional similarities to other dsRNA-binding proteins, including a mammalian nuclear protein that binds the human immunodeficiency virus (HIV) TAR element, and a mammalian dsRNase that is induced by interferon as part of the antiviral response. Understanding RNase III processing catalysis can provide a basis to define the role of protein-dsRNA interactions in human health and disease.