Fedor M J
Department of Molecular Biology, The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Biochemistry. 1999 Aug 24;38(34):11040-50. doi: 10.1021/bi991069q.
The hairpin ribozyme catalyzes a reversible RNA cleavage reaction that participates in processing intermediates of viral satellite RNA replication in plants. A minimal hairpin ribozyme consists of two helix-loop-helix segments. These segments associate noncoaxially in the active folded structure in a way that brings catalytically important loop nucleotides into close proximity. The hairpin ribozyme in the satellite RNA of Tobacco Ringspot Virus assembles in the context of a four-way helical junction. Recent physical characterization of hairpin ribozyme structures using fluorescence resonance energy transfer demonstrated enhanced stability of the folded structure in the context of a four-way helical junction compared to minimal hairpin ribozyme variants. Analysis of the functional consequences of this modification of the helical junction has revealed two changes in the hairpin ribozyme kinetic mechanism. First, ribozymes with a four-way helical junction bind 3' cleavage products with much higher affinity than minimal hairpin ribozymes, evidence that tertiary interactions within the folded structure contribute to product binding energy. Second, the balance between ligation and cleavage shifts in favor of ligation. The enhanced ligation activity of hairpin ribozymes that contain a four-way helical junction supports the notion that tertiary structure stability is a major determinant of the hairpin ribozyme proficiency as a ligase and illustrates the link between RNA structure and biological function.
发夹状核酶催化一种可逆的RNA切割反应，该反应参与植物中病毒卫星RNA复制中间体的加工过程。一个最小的发夹状核酶由两个螺旋-环-螺旋片段组成。这些片段在活性折叠结构中以非同轴方式结合，从而使具有催化重要性的环核苷酸紧密靠近。烟草环斑病毒卫星RNA中的发夹状核酶在一个四向螺旋连接的背景下组装。最近利用荧光共振能量转移对发夹状核酶结构进行的物理表征表明，与最小的发夹状核酶变体相比，在四向螺旋连接的背景下发夹状核酶折叠结构的稳定性增强。对这种螺旋连接修饰的功能后果分析揭示了发夹状核酶动力学机制的两个变化。第一，具有四向螺旋连接的核酶与3'切割产物的结合亲和力比最小的发夹状核酶高得多，这证明折叠结构内的三级相互作用有助于产物结合能。第二，连接和切割之间的平衡向有利于连接的方向转变。含有四向螺旋连接的发夹状核酶增强的连接活性支持了三级结构稳定性是发夹状核酶作为连接酶熟练程度的主要决定因素这一观点，并说明了RNA结构与生物学功能之间的联系。
