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。 远距离的 该项目的目标是确定 dsRNA 的酶促机制 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，一种参与 rRNA、tRNA 和 mRNA 的 dsRNA 特异性核酸酶 成熟、功能和衰退。 RNase III 包含一个结构域 特异性结合 dsRNA 以及第二个催化结构域 结合并位点特异性切割 dsRNA。 dsRNA 特异性核糖核酸酶 在所有检查的细胞中都发现了相似的结构域，并且执行相似的操作 职能角色。 已开发出 RNase III 作用模型 涉及 dsRNA 结合域和催化域的协调作用。 为了测试和完善该模型，具体目标是： 1. 确定如何 dsRNA 结合域通过催化作用增强底物裂解 域。 催化结构域、dsRNA 结合结构域、 有效切割所需的 dsRNA 底物将通过以下方式鉴定 特定蛋白质-dsRNA 的交联、保护和干扰测定 复合物。 2. 鉴定 dsRNA 必需的催化结构域成分 分裂。 催化中保守的、带电的或极性的氨基酸 结构域将发生突变，突变体酶裂解活性通过以下方法测量 动力学测定。 dsRNA 结合和切割活性 基因选择的 RNase III 突变体（由合作者 R. W. 西蒙斯）也将被测量。 所需金属离子的最少数量 裂解步骤将由动力学和平衡结合决定 实验。 3. 识别直接参与的 dsRNA 结合域组件 在 dsRNA 识别中。 假定识别中的保守残基 dsRNA 结合结构域的表面将发生突变，并且 dsRNA 结合结构域将发生突变。 和通过凝胶迁移率变化和过滤器结合测量的裂解活性 化验。 通过遗传筛选获得的突变体的 dsRNA 结合活性 将进行类似的测量。 破坏功能相互作用的突变 dsRNA 结合域和催化域之间的相互作用将被识别并 特点。

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.