Structure-Function Studies of Ribozyme and RNA

这一建议将继续锤头状核酶的结构和功能研究。锤头人 RIBO/YME已被证明在体外和体内都能有效地切割特定的RNA靶标。因此，这些 核酶在切割特定基因功能失活方面具有潜在的生物医学应用价值。 核糖核酸或作为抗病毒药物通过切割RNA病毒的基因组。这份提案中的工作重点是 对锤头状核酶的结构和动力学进行了详细的研究，主要是通过多种方法 三维异核核磁共振技术。最近的研究表明， 自然产生的植物类病毒锤头状核酶结构域稍大，切割速度要快得多 速度(-100倍于以前的锤头系统)。因此，这一次的主要焦点是 该提案将探索这些更快的锤头状核酶的结构和动力学。同位素 用T7RNA聚合酶和多种不同类型的核酶体外转录，可制备‘3C/’5N标记的核酶。 将进行多维异核核磁共振实验，以进行共振指认和 获取锤头状核酶的结构信息。核Overhauser效应、J偶合常数 将收集各种锤头结构上的剩余偶极耦合数据，并用于确定 这些核酶在溶液中的局部和整体结构。异核~(13)C核磁共振弛豫实验 将被用来测量这些核酶的动力学。这些松弛实验提供了信息 关于RNA中单个残基的构象动力学，以及与探针交换过程 寿命从微秒到毫秒不等。 将使用系综和单分子荧光共振能量转移技术来 研究锤头的三级相互作用和动力学。更快的一个耐人寻味的特性之一 裂解锤头是因为它的活性需要低得多的二价金属离子(Mg2)。两者都有 核磁共振和荧光技术将被用来探索镁对结构和动力学的影响 这些切割速度更快的锤头状核酶。

This proposal will continue structure-function studies on the hammerhead ribozyme. The hammerhead ribo/yme has been shown to efficiently cleave specific RNA targets both in vitro and in vivo. Thus these ribozymes have potential biomedical applications for inactivation of specific gene functions by cleavage of mRNA or as antiviral agents by cleavage of the genome of RNA viruses. The work in this proposal focuses on detailed studies of the structure and dynamics of the hammerhead ribozyme, primarily by multi- dimensional heteronuclear nuclear magnetic resonance (NMR) techniques. Recent studies have shown that a slightly larger domain of naturally occurring plant viroid hammerhead ribozymes has a much faster cleavage rate (-100 fold faster) than the previously characterized hammerhead systems. Thus the main focus of this proposal will be probing the structure and dynamics of these faster hammerhead ribozymes. Isotopically '3C/'5N-labeled ribozymes will be prepared by in vitro transcription with T7 RNA polymerase and a variety of multi-dimensional heteronuclear NMR experiments will be performed to make resonance assignmentand obtain structural information on the hammerhead ribozyme. Nuclear Overhauser effect, J-couplingconstants and residual dipolar couplings data will be collected on various hammerhead constructs and used to determine the local and global structure of these ribozymes in solution. Heteronuclear 13C NMR relaxationexperiments will be performed to measure dynamics on these ribozymes. These relaxation experiments give information on the conformational dynamics of individual residues in the RNA, and probe exchange processes with lifetimes from micro- to milliseconds. Both ensemble and single molecule fluorescence resonance energy transfer techniques will be used to study tertiary interactions and dynamics for the hammerhead. One of the intriguing properties of the faster cleaving hammerhead is that it requires much lower levels of divalent metal ions (Mg2+) for activity. Both NMR and fluorescence techniques will be used to probe the effect of Mg2+ on the structure and dynamicsof these faster cleaving hammerhead ribozymes.