REACTIVITY EPITOPES FOR RIBONUCLEASE III SUBSTRATES

核糖核酸酶 III 底物的反应性表位

• 批准号: 2841674
• 负责人: Allen Whitall Nicholson
• 金额: $ 20.83万
• 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 2003-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2841674
• 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的定点切割是细胞和病毒RNA成熟、功能和腐烂的关键步骤。核酸酶类似于核糖核酸酶III，存在于真核细胞中，具有相似的功能。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、功能和腐烂中的作用。特定的Watson-Crick碱基对序列决定了RNaseIII的结合位点，内部环决定了RNA链是一条还是两条被切割，切割模式反过来控制着RNA的功能和半衰期。具体目的是：1.确定与RNaseIII特异结合的Watson-Crick碱基对序列特征。RNaseIII对底物的识别依赖于裂解位点附近的特定Watson-Crick碱基对(W-CBP)序列。为了确定赋予特定结合的W-C BP序列特征，将测试含有BP取代基或缺乏官能团的碱基类似物的底物的体外结合和切割活性。在体外遗传选择将决定赋予特定结合的W-C碱基序列的范围。2.确定导致单链切割的RNA结构特征。内部环将双链切割模式切换为单链切割。为了确定这个基序如何允许RNaseIII结合，但提供单链切割，将测试具有改变的内部环序列的突变底物的结合亲和力和切割反应活性。在体外遗传选择将被用来确定赋予结合和/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‘-羟基和磷酸二酯氧基可能直接与RNaseIII接触并贡献总结合能。为了确定对结合2‘-脱氧硫代磷酸很重要的2’-羟基，将在修饰干扰试验中使用2‘-脱氧硫代取代的RNA，并将测试2’-脱氧取代的底物的体外结合和切割反应活性。离子和疏水对结合的贡献将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。