Nagy P D, Zhang C, Simon A E
Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003, USA.
EMBO J. 1998 Apr 15;17(8):2392-403. doi: 10.1093/emboj/17.8.2392.
Molecular mechanisms of RNA recombination were studied in turnip crinkle carmovirus (TCV), which has a uniquely high recombination frequency and non-random crossover site distribution among the recombining TCV-associated satellite RNAs. To test the previously proposed replicase-driven template-switching mechanism for recombination, a partially purified TCV replicase preparation (RdRp) was programed with RNAs resembling the putative in vivo recombination intermediates. Analysis of the in vitro RdRp products revealed efficient generation of 3'-terminal extension products. Initiation of 3'-terminal extension occurred at or close to the base of a hairpin that was a recombination hotspot in vivo. Efficient generation of the 3'-terminal extension products depended on two factors: (i) a hairpin structure in the acceptor RNA region and (ii) a short base-paired region formed between the acceptor RNA and the nascent RNA synthesized from the donor RNA template. The hairpin structure bound to the RdRp, and thus is probably involved in its recruitment. The probable role of the base-paired region is to hold the 3' terminus near the RdRp bound to the hairpin structure to facilitate 3'-terminal extension. These regions were also required for in vivo RNA recombination between TCV-associated sat-RNA C and sat-RNA D, giving crucial and direct support for a replicase-driven template-switching mechanism of RNA recombination.
对芜菁皱缩病毒（TCV）的RNA重组分子机制进行了研究，该病毒在重组的TCV相关卫星RNA中具有独特的高重组频率和非随机交叉位点分布。为了测试先前提出的复制酶驱动的模板切换重组机制，用类似于假定的体内重组中间体的RNA对部分纯化的TCV复制酶制剂（RdRp）进行编程。对体外RdRp产物的分析揭示了3'末端延伸产物的有效产生。3'末端延伸的起始发生在体内作为重组热点的发夹底部或其附近。3'末端延伸产物的有效产生取决于两个因素：（i）受体RNA区域中的发夹结构，以及（ii）受体RNA与从供体RNA模板合成的新生RNA之间形成的短碱基配对区域。发夹结构与RdRp结合，因此可能参与其募集。碱基配对区域的可能作用是将3'末端保持在与发夹结构结合的RdRp附近，以促进3'末端延伸。这些区域对于TCV相关的卫星RNA C和卫星RNA D之间的体内RNA重组也是必需的，为RNA重组的复制酶驱动的模板切换机制提供了关键且直接的支持。
