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首页> 外文期刊>Journal of Molecular Biology >Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia.
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Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia.

机译：贾第鞭毛虫转录本的衣壳编码区域中的特定二级结构是其在贾第鞭毛虫中的翻译所必需的。

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摘要

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giardia lamblia requires the presence of the initial 264 nucleotides of the viral capsid-coding region. A 13 nt downstream box (DB) sequence within this region, complementary to a 15 nt sequence near the 3' end of G. lamblia 16 S-like ribosomal RNA (rRNA), was found to be essential for the enhanced translation. However, DB is located 64-78 nt downstream of the initiation codon, whereas an exponential increase of translation efficiency depends on a further increment of the coding region from nucleotides 111 to 264. Thus, there could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designated I to IV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (DeltaG degrees ) of -77.16 kcal x mol(-1). Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-loops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem-loop IVA at a nearby location in the molecule. Site-directed mutagenesis designed to disrupt stem-loop structures I, II, III or IVA resulted in drastic reduction of translation efficiency, which was restored by compensatory sequence changes to regenerate individual stem-loop structures. Mutations disrupting the originally designated stem-loop IV did not exert any detectable effect on translation. However, alterations of the sequence UCUCC between nucleotides 216 and 220 in the flexible loop region of the revised secondary structure led to a precipitous drop in translation. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experimental support. Changes of the DB sequence without affecting the postulated stem structure led to drastic losses of translation efficiency. Thus, a simple structural basis for the enhanced translation could be that stem-loops I, II, III and IVA and the UCUCC sequence may facilitate the interaction between DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia. Copyright 2001 Academic Press.
机译：贾第鞭毛虫中贾第鞭毛虫病毒（GLV）-荧光素酶嵌合mRNA的增强翻译需要存在病毒衣壳编码区的起始264个核苷酸。发现该区域内的一个13 nt下游盒（DB）序列与兰伯氏菌16 S样核糖体RNA（rRNA）3'端附近的一个15 nt序列互补，对增强翻译至关重要。但是，DB位于起始密码子的下游64-78 nt，而翻译效率的指数增长取决于编码区从核苷酸111到264的进一步增加。因此，可能需要对结构进行额外的结构增强DB下游的区域。在264 nt衣壳编码区的MFOLD预测二级结构中鉴定出四个主要的茎-环结构，命名为I至IV，估计的最小自由能（DeltaG度）为-77.16 kcal x mol（-1）。我们对溶液中游离264 nt RNA分子的化学探测分析支持了茎环I，II和III的预测存在，但对茎环IV产生了怀疑。相反，这表明在分子的附近位置存在茎环IVA。设计用于破坏茎环结构I，II，III或IVA的定点诱变导致翻译效率急剧降低，可通过补偿序列变化来恢复单个茎环结构，从而恢复翻译效率。破坏最初指定的茎环IV的突变对翻译没有任何可检测的作用。然而，在修饰的二级结构的柔性环区域中核苷酸216和220之间的序列UCUCC的改变导致翻译的急剧下降。 MFOLD预测的另一个茎环，由DB序列的主要部分组成，已通过化学探测进行了检查，但几乎没有实验支持。在不影响假定的茎结构的情况下改变DB序列会导致翻译效率的巨大损失。因此，增强翻译的简单结构基础可能是茎环I，II，III和IVA和UCUCC序列可以促进DB和抗DB之间16 S样rRNA中的相互作用，从而启动GLV mRNA的翻译。在G. lamblia。版权所有2001学术出版社。

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