Pan T, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309.
Biochemistry. 1992 Apr 28;31(16):3887-95. doi: 10.1021/bi00131a001.
An in vitro selection method has been developed to obtain RNA molecules that specifically undergo autolytic cleavage reactions by Pb2+ ion. The method utilizes a circular RNA intermediate which is regenerated following the cleavage reaction to allow amplification and multiple cycles of selection. Pb2+ is known to catalyze a specific cleavage reaction between U17 and G18 of yeast tRNA(Phe). Starting from pools of RNA molecules which have a random distribution of sequences at nine or ten selected positions in the sequence of yeast tRNA(Phe), we have isolated many RNA molecules that undergo rapid and specific self-cleavage with Pb2+ at a variety of different sites. Terminal truncation experiments suggest that most of these self-cleaving RNA molecules do not fold like tRNA. However, two of the variants are cleaved rapidly with Pb2+ at U17 even though they lack the highly conserved nucleotides G18 and G19. Both specific mutations and terminal truncation experiments suggest that the D and T loops of these two variants interact in a manner similar to that of tRNA(Phe) despite the absence of the G18U55 and G19C56 tertiary interactions. A model for an alternate tertiary interaction involving a U17U55 pair is presented. This model may be relevant to the structure of about 100 mitochondrial tRNAs that also lack G18 and G19. The selection method presented here can be directly applied to isolate catalytic RNAs that undergo cleavage in the presence of other metal ions, modified nucleotides, or sequence-specific nucleases.
已开发出一种体外筛选方法，以获得能通过Pb2+离子特异性进行自溶裂解反应的RNA分子。该方法利用一种环状RNA中间体，其在裂解反应后会再生，以允许扩增和多轮筛选。已知Pb2+催化酵母tRNA（Phe）的U17和G18之间的特异性裂解反应。从在酵母tRNA（Phe）序列中九个或十个选定位置具有随机序列分布的RNA分子库开始，我们已分离出许多能在各种不同位点与Pb2+发生快速且特异性自裂解的RNA分子。末端截短实验表明，这些自裂解RNA分子中的大多数折叠方式与tRNA不同。然而，其中两个变体即使缺乏高度保守的核苷酸G18和G19，也能在U17处被Pb2+快速裂解。特异性突变和末端截短实验均表明，尽管缺乏G18U55和G19C56三级相互作用，但这两个变体的D环和T环仍以与tRNA（Phe）相似的方式相互作用。提出了一种涉及U17U55对的替代三级相互作用模型。该模型可能与约100种也缺乏G18和G19的线粒体tRNA的结构相关。本文介绍的筛选方法可直接应用于分离在其他金属离子、修饰核苷酸或序列特异性核酸酶存在下发生裂解的催化RNA。
