Feldstein P A, Bruening G
Department of Plant Pathology, College of Agricultural and Environmental Sciences, University of California, Davis 95616.
Nucleic Acids Res. 1993 Apr 25;21(8):1991-8. doi: 10.1093/nar/21.8.1991.
The less abundant polarity of the satellite RNA of tobacco ringspot virus, designated sTobRV(-)RNA, contains a ribozyme and its substrate. We demonstrate that the ribozyme can catalyze the ligation of substrate cleavage products and that oligoribonucleotides, termed 'mini-monomers' and containing little more than covalently attached ribozyme and substrate cleavage products, circularized spontaneously, efficiently and reversibly. The kinetics of ligation and cleavage of one such mini-monomer was consistent with a simple unimolecular reaction at some temperatures. Evidence suggests that the circular ligation product includes a 5 bp stem that is connected to a 4 bp stem by a bulge loop. Reduction of the bulge loop to one nt is expected to place the 4 and 5 bp helices in a nearly coaxial, rather than an angled or parallel, orientation. Such molecules did not circularize in a unimolecular reaction but did when incubated with second, trans-acting oligoribonucleotides that had either the original or a substituted 4 bp helix. These results suggest that a bulge loop that is too small prevents formation of geometry essential for unimolecular ligation. We suggest the term 'paperclip' to represent the arrangement of RNA strands in the region of sTobRV(-)RNA that participates in the cleavage and ligation reactions.
烟草环斑病毒卫星RNA中丰度较低的负极性链，即sTobRV(-)RNA，含有一种核酶及其底物。我们证明该核酶能够催化底物切割产物的连接，并且一种被称为“迷你单体”的寡核糖核苷酸，其包含的共价连接的核酶和底物切割产物仅比其他成分略多，能自发、高效且可逆地环化。在某些温度下，一种此类迷你单体的连接和切割动力学与简单的单分子反应一致。有证据表明，环状连接产物包括一个5碱基对的茎，该茎通过一个凸起环与一个4碱基对的茎相连。将凸起环减少至一个核苷酸预计会使4碱基对和5碱基对的螺旋处于几乎同轴而非成角度或平行的方向。此类分子在单分子反应中不会环化，但与具有原始或替代的4碱基对螺旋的第二条反式作用寡核糖核苷酸一起孵育时会环化。这些结果表明，过小的凸起环会阻止单分子连接所需的几何结构形成。我们建议用“回形针”一词来表示sTobRV(-)RNA中参与切割和连接反应区域的RNA链排列。
