Dominguez D I, Ryabova L A, Pooggin M M, Schmidt-Puchta W, Fütterer J, Hohn T
Friedrich-Miescher-Institute, CH-4002 Basel, Switzerland.
J Biol Chem. 1998 Feb 6;273(6):3669-78. doi: 10.1074/jbc.273.6.3669.
A wheat germ cell-free system was used to study details of ribosome shunting promoted by the cauliflower mosaic virus 35 S RNA leader. By testing a dicistronic construct with the leader placed between two coding regions, we confirmed that the 35 S RNA leader does not include an internal ribosome entry site of the type observed with picornavirus RNAs. A reporter gene fused to the leader was shown to be expressed by ribosomes that had followed the bypass route (shunted) and, with lower efficiency, by ribosomes that had scanned through the whole region. Stem section 1, the most stable of the three stem sections of the leader, was shown to be an important structural element for shunting. Mutations that abolished formation of this stem section drastically reduced reporter gene expression, whereas complementary mutations that restored stem section 1 also restored shunting. A micro-leader capable of shunting consisting of stem section 1 and flanking sequences could be defined. A small open reading frame preceding stem section 1 enhances shunting.
利用小麦胚无细胞体系研究花椰菜花叶病毒35S RNA前导序列促进核糖体跳跃的细节。通过测试一个双顺反子构建体（前导序列位于两个编码区之间），我们证实35S RNA前导序列不包含与微小核糖核酸病毒RNA中观察到的类型相同的内部核糖体进入位点。与前导序列融合的报告基因显示，通过旁路途径（跳跃）的核糖体能够表达该基因，而通过扫描整个区域的核糖体表达效率较低。前导序列的三个茎环结构中最稳定的茎环结构1，被证明是跳跃的一个重要结构元件。消除该茎环结构形成的突变会大幅降低报告基因的表达，而恢复茎环结构1的互补突变也能恢复跳跃。可以定义一个由茎环结构1及其侧翼序列组成的能够跳跃的微型前导序列。茎环结构1之前的一个小开放阅读框可增强跳跃。
