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成熟、功能和降解的关键步骤。 类似于RNase III的核酸酶存在于真核细胞中，并且执行类似的功能作用。 长期目标是确定双链RNA识别和切割的酶机制及其在RNA成熟，功能和衰变中的作用。 特异性的沃森-克里克碱基对序列决定了RNA酶III的结合位点，而内环决定了一条或两条RNA链是否被切割。切割的模式反过来控制RNA的功能和半衰期。 具体目标是：1. 确定赋予RNase III特异性结合的沃森-克里克碱基对序列特征。 RNase III对底物的识别依赖于切割位点附近的特异性Watson-Crick碱基对（W-C bp）序列。 为了确定赋予特异性结合的W-C bp序列特征，将检测含有bp取代或缺少官能团的碱基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异性结合的W-C bp序列的范围。 2. 确定赋予单链切割的RNA结构特征。 内环将双链切割模式转换为单链切割模式。 为了确定该基序如何允许RNA酶III结合，但赋予单链切割，将测试具有改变的内环序列的突变体底物的结合亲和力和切割反应性。 体外遗传选择将用于确定赋予结合和/或切割的内环结构的范围。 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 '-羟基和磷酸二酯氧可以直接接触RNA酶III并有助于总结合能。 为了鉴定对于结合重要的2 '-羟基，将在修饰-干扰测定中使用2'-脱氧硫代磷酸酯取代的RNA，并且将测试定点2 '-脱氧取代的底物的体外结合和切割反应性。 通过测量底物结合亲和力分别作为盐浓度和温度的函数来确定离子和疏水性对结合的贡献。