Dulude Dominic, Baril Martin, Brakier-Gingras Léa
Département de Biochimie, Université de Montréal, 2900 Boulevard Edouard-Montpetit, Montréal, Québec H3T 1J4, Canada.
Nucleic Acids Res. 2002 Dec 1;30(23):5094-102. doi: 10.1093/nar/gkf657.
Synthesis of the Gag-Pol protein of the human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshifting when ribosomes translate the unspliced viral messenger RNA. This frameshift occurs at a slippery sequence followed by an RNA structure motif that stimulates frameshifting. This motif is commonly assumed to be a simple stem-loop for HIV-1. In this study, we show that the frameshift stimulatory signal is more complex than believed and consists of a two-stem helix. The upper stem-loop corresponds to the classic stem-loop, and the lower stem is formed by pairing the spacer region following the slippery sequence and preceding this classic stem-loop with a segment downstream of this stem-loop. A three-purine bulge interrupts the two stems. This structure was suggested by enzymatic probing with nuclease V1 of an RNA fragment corresponding to the gag/pol frameshift region of HIV-1. The involvement of the novel lower stem in frameshifting was supported by site-directed mutagenesis. A fragment encompassing the gag/pol frameshift region of HIV-1 was inserted in the beginning of the coding sequence of a reporter gene coding for the firefly luciferase, such that expression of luciferase requires a -1 frameshift. When the reporter was expressed in COS cells, mutations that disrupt the capacity to form the lower stem reduced frameshifting, whereas compensatory changes that allow re-formation of this stem restored the frameshift efficiency near wild-type level. The two-stem structure that we propose for the frameshift stimulatory signal of HIV-1 differs from the RNA triple helix structure recently proposed.
当核糖体翻译未剪接的人类免疫缺陷病毒1型（HIV-1）信使核糖核酸时，HIV-1的Gag-Pol蛋白合成需要程序性-1核糖体移码。这种移码发生在一个滑序列之后，接着是一个刺激移码的RNA结构基序。通常认为HIV-1的这个基序是一个简单的茎环结构。在本研究中，我们表明移码刺激信号比认为的更复杂，由一个双茎螺旋组成。上部茎环对应于经典茎环，下部茎是由滑序列之后、经典茎环之前的间隔区与该茎环下游的一段序列配对形成的。一个三嘌呤凸起中断了这两个茎。这种结构是通过用核酸酶V1对对应于HIV-1 gag/pol移码区的RNA片段进行酶切探测而提出的。定点诱变支持了新的下部茎在移码中的作用。将包含HIV-1 gag/pol移码区的片段插入到编码萤火虫荧光素酶的报告基因编码序列的开头，使得荧光素酶的表达需要-1移码。当报告基因在COS细胞中表达时，破坏形成下部茎能力的突变会降低移码，而允许该茎重新形成的补偿性变化会使移码效率恢复到接近野生型水平。我们提出的HIV-1移码刺激信号的双茎结构不同于最近提出的RNA三螺旋结构。
