IDENTIFICATION OF THE MOTIFS WITHIN THE TOBACCO MOSAIC-VIRUS 5'-LEADER RESPONSIBLE FOR ENHANCING TRANSLATION
IDENTIFICATION OF THE MOTIFS WITHIN THE TOBACCO MOSAIC-VIRUS 5'-LEADER RESPONSIBLE FOR ENHANCING TRANSLATION
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DOI：
10.1093/nar/20.17.4631
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发表时间：
1992-09-11
影响因子：
14.9
通讯作者：
WALBOT, V
中科院分区：
文献类型：
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作者：
GALLIE, DR;WALBOT, V
烟草花叶病毒RNA的前导序列（称为OMEGA）在真核生物和原核生物中都能增强翻译。虽然预测在OMEGA内存在很少的二级结构，但68个碱基的前导序列的一级序列是高度有组织的。三个拷贝的八个碱基的同向重复序列和（CAA）n区域代表在许多TMV株系的前导序列中发现的两个基序，并且它们一起构成OMEGA的72%。在先前的缺失研究中，没有突变体表现出功能丧失，表明OMEGA内存在功能冗余。我们在这里报告，一个更全面的缺失分析确定的图案参与翻译增强。在另一种方法中，使用含有每个基序序列的寡核苷酸来构建基序数量和构型不同的前导序列。在体外合成含有这些突变前导序列的β-葡糖醛酸酶mRNA构建体，并在通过电穿孔将mRNA递送至胡萝卜原生质体后在体内测量其翻译效率。一个拷贝的8个碱基的直接重复序列和一个25个碱基的（CAA）n区域的组合被鉴定为核心调控元件，尽管（CAA）n基序更关键。两个拷贝的（CAA）n区域足以赋予高水平的增强，并且由多个拷贝的直接重复组成的前导序列是中度增强的。因此，这两个基序在功能上是冗余的。
The leader (called OMEGA) of tobacco mosaic virus RNA enhances translation in both eukaryotes and prokaryotes. Although little secondary structure is predicted to exist within OMEGA, the primary sequence of the 68 base leader is highly organized. Three copies of an eight base direct repeat and a (CAA)n region represent the two motifs found in the leaders of many TMV strains, and together these comprise 72% of OMEGA. In previous deletion studies, no mutants exhibited loss-of-function, suggesting that functional redundancy exists within OMEGA. We report here that a more comprehensive deletion analysis identified the motifs involved in translational enhancement. In a separate approach, oligonucleotides containing the sequence of each motif were used to construct leaders that varied in the number and configuration of the motifs. beta-Glucuronidase mRNA constructs containing these mutant leaders were synthesized in vitro and their translational efficiency measured in vivo following mRNA delivery to carrot protoplasts via electroporation. A combination of one copy of the 8 base direct repeat and a 25 base (CAA)n region was identified as the core regulatory element, although the (CAA)n motif is more critical. Two copies of the (CAA)n region are sufficient to confer a high level of enhancement and a leader composed of multiple copies of the direct repeat is moderately enhancing. Thus, these two motifs are functionally redundant.