Garlapati S, Chou J, Wang C C
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-0446, USA.
J Mol Biol. 2001 May 11;308(4):623-38. doi: 10.1006/jmbi.2001.4568.
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.
贾第虫病毒（GLV）-荧光素酶嵌合mRNA在蓝氏贾第鞭毛虫中的增强翻译需要病毒衣壳编码区最初的264个核苷酸的存在。该区域内一个13个核苷酸的下游框（DB）序列，与蓝氏贾第鞭毛虫16S样核糖体RNA（rRNA）3'端附近的一个15个核苷酸序列互补，被发现对增强翻译至关重要。然而，DB位于起始密码子下游64-78个核苷酸处，而翻译效率的指数增加取决于编码区从第111个核苷酸到第264个核苷酸的进一步增加。因此，DB下游区域的翻译增强可能存在额外的结构要求。在264个核苷酸的衣壳编码区的MFOLD预测二级结构中鉴定出四个主要的茎环结构，分别命名为I至IV，估计最小自由能（ΔG°）为-77.16千卡×摩尔-1。我们对溶液中游离的264个核苷酸RNA分子的化学探针分析支持茎环I、II和III的预测存在，但对茎环IV表示怀疑。相反，它表明在分子的附近位置存在一个茎环IVA。旨在破坏茎环结构I、II、III或IVA的定点诱变导致翻译效率急剧降低，通过补偿性序列变化恢复单个茎环结构可使其恢复。破坏最初指定的茎环IV的突变对翻译没有任何可检测到的影响。然而，在修订后的二级结构的柔性环区域中，第216至220个核苷酸之间的UCUCC序列的改变导致翻译急剧下降。MFOLD预测的另一个由DB序列的主要部分组成的茎环通过化学探针进行了检测，但几乎没有得到实验支持。不影响假定茎结构的DB序列变化导致翻译效率急剧损失。因此，增强翻译的一个简单结构基础可能是茎环I、II、III和IVA以及UCUCC序列可能促进DB与16S样rRNA中的反DB之间的相互作用，从而启动蓝氏贾第鞭毛虫中GLV mRNA的翻译。
