RNASE III MECHANISM OF DOUBLE-STRANDED RNA CLEAVAGE

描述(改编自申请人的摘要)：认可和 双螺旋RNA的切割对于细胞的成熟、功能、 以及细胞和病毒RNA分子的衰变。这些过程是 由双链(DS)-RNA特异性核糖核酸酶催化。长距离的 本项目的目标是确定dsrna的酶机制。 裂解及其在调节细胞和病毒RNA代谢中的作用 功能。短期目标是分析大肠杆菌核糖核酸酶 III，一种dsRNA特异的核酸酶，参与rRNA、tRNA和mRNA. 成熟、功能和腐烂。RNaseIII包含一个域，它 特异性地结合dsrna，以及第二个催化结构域，它可以 结合并定点切割dsRNA。DsRNA特异性核糖核酸酶 在所有被检查的细胞中发现相似的结构域，并且执行类似的操作 职能角色。一个核糖核酸酶III的作用模型已经开发出来 涉及dsRNA结合域和催化域的协调作用。 测试和改进此模型的具体目标是：1.确定如何 DsRNA结合域通过催化作用增强底物切割 域。催化结构域、dsRNA结合域、 和有效切割所需的dsRNA底物将通过 特异性蛋白质dsRNA的交联、保护和干扰分析 复合体。2.确定dsRNA必需的催化结构域成分 乳沟。催化剂中的保守、带电或极性氨基酸 结构域将发生突变，突变的酶切割活性将通过 动力学分析。的dsRNA结合和切割活性 基因选择的RNaseIII突变体(合作者R.W. 西蒙斯)也将被衡量。所需金属离子的最小数量 因为切割步骤将由动力学和平衡结合决定 实验。3.确定直接参与的dsRNA结合域组件 在dsRNA识别中。推定识别中的保守残基 DsRNA结合域的表面会发生突变，并与dsRNA结合 并通过凝胶迁移率变化和过滤器结合来测量切割活性 化验。遗传筛选获得的突变体的dsRNA结合活性 也会有类似的衡量标准。扰乱功能相互作用的突变 DsRNA结合域和催化域之间的关系将被确定并 特色化的。

DESCRIPTION (Adapted from applicant's abstract): The recognition and cleavage of double-helical RNA is essential for the maturation, function, and decay of cellular and viral RNA molecules. These processes are catalyzed by double-stranded(ds)-RNA-specific ribonucleases. The long-range goal of this project is to determine the enzymatic mechanism of dsRNA cleavage and its role in regulating cellular and viral RNA metabolism and function. The short-term goal is to analyze Escherichia coli ribonuclease III, a dsRNA-specific nuclease that participates in rRNA, tRNA and mRNA maturation, function and decay. RNase III contains a domain that specifically binds dsRNA, as well as a second, catalytic domain which can bind and site-specifically cut dsRNA. dsRNA-specific ribonucleases with similar domains are found in all cells examined, and perform similar functional roles. A model for RNase III action has been developed that involves the coordinated action of the dsRNA-binding and catalytic domains. To test and refine this model the specific aims are to: 1. Determine how the dsRNA-binding domain enhances substrate cleavage by the catalytic domain. Interactions between the catalytic domain, dsRNA-binding domain, and dsRNA substrate required for efficient cleavage will be identified by crosslinking, protection and interference assays on specific protein-dsRNA complexes. 2. Identify catalytic domain components essential for dsRNA cleavage. The conserved, charged or polar amino acids in the catalytic domain will be mutated and mutant enzyme cleavage activities measured by kinetic assays. The dsRNA-binding and cleavage activities of genetically-selected RNase III mutants (provided by collaborator R. W. Simons) also will be measured. The minimum number of metal ions required for the cleavage step will be determined by kinetic and equilibrium binding experiments. 3. Identify dsRNA-binding domain components directly involved in dsRNA recognition. Conserved residues in the putative recognition surface of the dsRNA-binding domain will be mutated and the dsRNA-binding and cleavage activities measured by gel mobility shift and filter binding assays. The dsRNA-binding activities of mutants obtained by genetic screens will be similarly measured. Mutations which disrupt functional interactions between the dsRNA-binding and catalytic domains will be identified and characterized.