Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.
Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.
复制标题
苜蓿花叶病毒外壳蛋白 mRNA 3 非翻译区竞争性翻译决定簇的鉴定。
DOI：
10.1128/mcb.17.4.2005
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发表时间：
1997
影响因子：
5.3
通讯作者：
Gehrke,L
中科院分区：
文献类型：
--
作者：
Hann,LE;Webb,AC;Cai,JM;Gehrke,L
苜蓿花叶病毒外壳蛋白（CP RNA）是一种非腺苷化的mRNA，其竞争翻译活性部分由3′非翻译区（UTR）决定。竞争性翻译的特点是在体外，与共翻译测定，并在体内，与microinjectedXenopus laevisoocytes。在小麦胚芽提取物中，外壳蛋白的合成是恒定的，当一个固定量的全长CP RNA与竞争珠蛋白mRNA的浓度增加共翻译。然而，在竞争条件下，缺少3′ UTR的CP RNA的翻译显著下降。RNA稳定性相当。InX。在哺乳动物细胞中，这是一个饱和的，是一个固有的竞争性翻译环境，全长CP RNA组装成大的多聚核糖体和外壳蛋白的合成是容易检测的。或者，缺少3′ UTR的CP RNA沉淀为小的多聚核糖体，并且检测到很少的外壳蛋白。同样，RNA稳定性是等同的。定点诱变用于定位竞争性翻译所需的RNA序列或结构。由于CP RNA 3′ UTR中有大量的AUG核苷酸三联体，因此在卵母细胞注射前，全长RNA中的两个含AUG的位点发生了改变。在外壳蛋白终止密码子下游20个核苷酸的序列GAUG处的核苷酸取代特异性地减少全长CP RNA翻译，而在下一个AUG三联体处的类似取代对翻译几乎没有影响。3′ UTR的竞争性影响可以通过影响翻译起始的RNA-蛋白质相互作用或通过下游AUG密码子处的核糖体再起始来解释，这将增加用于外壳蛋白合成的核糖体的数量。
We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3′ untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjectedXenopus laevisoocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3′ UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. InX. laevisoocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3′ UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3′ UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3′ UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.