REACTIVITY EPITOPES FOR RIBONUCLEASE III SUBSTRATES

核糖核酸酶 III 底物的反应性表位

• 批准号: 6519844
• 负责人: Allen Whitall Nicholson
• 金额: $ 17.04万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519844
• 关键词: chemical binding; chemical cleavage; conformation; double stranded RNA; enzyme mechanism; enzyme substrate; enzyme substrate complex; gel mobility shift assay; hydropathy; hydroxyl group; ionic bond; nucleic acid sequence; nucleic acid structure; polymerase chain reaction; ribonuclease III; site directed mutagenesis; thermodynamics

The site-specific cleavage of double-stranded RNA by ribonuclease III of Escherichia coli is a key step in the maturation, function and decay of cellular and viral RNAs. Nucleases similar to RNase III occur in eukaryotic cells, and perform similar functional roles. The long-term objective is to determine the enzymatic mechanism of double-stranded RNA recognition and cleavage and its role in RNA maturation, function and decay. Specific Watson- Crick base-pair sequences determine the RNase III binding site, and an internal loop determines whether one or both RNA strands are cleaved The pattern of cleavage in turn controls RNA function and half-life. The Specific Aims are to: 1. Determine the Watson-Crick base-pair sequence features which confer specific binding of RNase III. RNase III recognition of substrate is dependent upon specific Watson-Crick base-pair (W-C bp) sequences near the cleavage site. To determine W-C bp sequence features which confer specific binding, substrates containing bp substitutions or base analogues lacking functional groups will be tested for their in vitro binding and cleavage activities. In vitro genetic selection will determine the range of W-C bp sequences that confers specific binding. 2. Determine the RNA structural features that confer single-strand cleavage. An internal loop switches the pattern of double-strand cleavage to single-strand cleavage. To determine how this motif allows RNase III binding, but confers single-strand cleavage, mutant substrates with altered internal loop sequences will be tested for their binding affinities and cleavage reactivities. In vitro genetic selection will be used to determine the range of internal loop structures that confer binding and/or cleavage. 3. Determine 2'-hydroxyl group involvement in binding, and the energetic contribution of ionic and hydrophobic interactions. A specific set of substrate 2'-hydroxyls and phosphodiester oxygens may directly contact RNase III and contribute to overall binding energy. To identify the 2'-hydroxyl groups important for binding 2'-deoxyphosphorothioate-substituted RNAs will be used in modification-interference assays, and site-directed 2'-deoxy- substituted substrates will be tested for their in vitro binding and cleavage reactivities. The ionic and hydrophobic contributions to binding will be determined by measuring substrate binding affinities as a function of salt concentration and temperature, respectively.

大肠杆菌的核糖核酸酶III对双链RNA的位点特异性切割是细胞和病毒RNA的成熟，功能和衰减的关键步骤。 与RNase III相似的核酸酶发生在真核细胞中，并发挥相似的功能作用。 长期目标是确定双链RNA识别和切割的酶促机制及其在RNA成熟，功能和衰减中的作用。 特定的watson-crick碱基对序列确定RNase III结合位点，并且内部环确定一个或两条RNA链是否被切割为裂解的裂解模式转基因控制RNA功能和半寿命。 具体目的是：1。确定赋予RNase III特定结合的Watson-Crick碱基对序列特征。 RNase III的底物识别取决于裂解位点附近的特定Watson-Crick碱基对（W-C BP）序列。 为了确定赋予特定结合的W-C BP序列特征，将测试包含BP取代或缺乏官能团体的基础类似物的底物的体外结合和切割活性。 体外遗传选择将确定赋予特定结合的W-C BP序列的范围。 2。确定赋予单链裂解的RNA结构特征。 内部环将双链裂解的模式切换到单链裂解。 为了确定该基序如何允许RNase III结合，但赋予单链裂解，将测试具有改变内部环序列的突变底物的结合亲和力和裂解反应率。 体外遗传选择将用于确定赋予结合和/或切割的内部环结构的范围。 3。确定2'-羟基参与结合，以及离子和疏水相互作用的能量贡献。 一组特定的底物2'-羟基和磷酸化酯氧气可以直接接触RNase III，并有助于总体结合能。 为了鉴定2'-羟基对结合2'-脱氧磷酸硫酸硫酸硫酸硫酸硫酸盐的取代的重要性RNA，将用于修饰干扰测定法中，将测试以2'-脱氧替代的底物测试其体外结合和裂解相反率。 离子和疏水对结合的贡献将通过分别测量底物结合亲和力作为盐浓度和温度的函数来确定。