Ribonuclease III mechanism of double-strand RNA cleavage

描述（由申请人提供）：双链RNA的酶促切割是许多真核和原核RNA成熟和衰变的重要步骤。双链（ds）RNA切割由核糖核酸内切酶的核糖核酸酶III超家族的成员进行。RNase III直系同源物参与mRNA和rRNA成熟、mRNA降解和反义RNA作用。最近的一个重要发现是RNase III直向同源物“Dicer”在RNA干扰（RNAi）中的中心参与。RNAi被认为可以抑制病毒感染，抑制逆转录酶运动，并参与真核生物（包括人类）的特定发育途径。高度保守的序列和结构元件的出现表明RNase III直系同源物使用相同的催化机制，这是未知的。核糖核酸酶Ⅲ超家族中研究最多的成员是大肠杆菌核糖核酸酶Ⅲ，最近在确定大肠杆菌核糖核酸酶Ⅲ方面取得了重大进展。螺旋RNA酶III的作用机制。本项目的长期目标是确定E.螺旋核糖核酸酶III。本项目的具体目标是：（1）确定催化结构域中保守氨基酸的功能作用。为了实现这一点，将在底物结合和切割测定中测试具有保守催化结构域残基取代的RNA酶III突变体;（2）确定二价金属离子化学计量和催化功能。为了实现这一点，将进行切割的动力学测定和使用RNase III和所选突变体的金属离子结合测定;（3）鉴定赋予结合能和优化催化效率的RNase III双链RNA结合结构域（dsRBD）中的序列和结构特征。这将通过分析特异性dsRBD突变体的RNA结合和通过噬菌体展示来实现;（4），确定RNA酶III-底物复合物的化学计量，并鉴定对RNA酶III功能重要的亚基间相互作用。将使用人工异源二聚体进行凝胶过滤、离心和底物结合和切割试验。实现这些目标不仅将为E.螺旋RNA酶III，而且还将提供关于RNA酶III直向同源物在真核RNA加工和降解途径（包括RNAi）中的功能的机制见解。

DESCRIPTION (provided by applicant): The enzymatic cleavage of double-stranded RNA is an essential step in the maturation and decay of many eukaryotic and prokaryotic RNAs. Double-stranded (ds) RNA cleavage is carried out by members of the ribonuclease Ill superfamily of endoribonucleases. RNase III orthologues participate in mRNA and rRNA maturation, mRNA degradation, and antisense RNA action. An important recent finding is the central involvement of the RNase III orthologue "Dicer" in RNA interference (RNAi). RNAi is proposed to suppress viral infection, inhibit retroposon movement, and participate in specific developmental pathways in eukaryotes, including humans. The occurrence of highly conserved sequences and structural elements indicate that RNase Ill orthologues use the same catalytic mechanism, which is unknown. The most studied member of the RNase III superfamily is Escherichia coli ribonuclease III and there has been recent significant progress in determining the E. coil RNase Ill mechanism of action. The long-range goal of this project is to determine the mechanism of dsRNA cleavage by E. coil RNase Ill. The specific aims of the project are to: (1) determine the functional roles of conserved amino acids in the catalytic domain. To accomplish this, RNase Ill mutants with substitutions of conserved catalytic domain residues will be tested in substrate binding and cleavage assays; (2), determine divalent metal ion stoichiometry and function in catalysis. To accomplish this, kinetic assays of cleavage, and metal ion binding assays using RNase Ill and selected mutants will be performed; (3), identify sequence and structural features in the RNase Ill double-stranded-RNA-binding domain (dsRBD) that confer binding energy and optimize catalytic efficiency. This will be accomplished by analyzing RNA binding of specific dsRBD mutants, and by phage display; (4), determine the stoichiometry of an RNase Ill-substrate complex, and identify intersubunit interactions important for RNase Ill function. Gel filtration, centrifugation, and substrate binding and cleavage assays using artificial heterodimers will be performed. Accomplishing these aims not only will provide important information on E. coil RNase Ill, but will also provide mechanistic insight on RNase Ill orthologue function in eukaryotic RNA processing and degradation pathways, including RNAi.