Specific secondary structures in the capsid-coding region of giardiavirus transcript are required for its translation in Giardia lamblia

Enhanced translation of giardiavirus (GLV)-luciferase chimeric mRNA in Giar dia lamblia requires the presence of the initial 264 nucleotides of the vir al capsid-coding region. A 13 nt downstream box (DB) sequence within this r egion, 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 tra nslation. However, DB is located 64-78 nt downstream of the initiation codo n, whereas an exponential increase of translation efficiency depends on a f urther increment of the coding region from nucleotides 111 to 264. Thus, th ere could be additional structural requirements for translation enhancement in the region downstream from DB. Four major stem-loop structures, designa ted I to TV, were identified in the MFOLD-predicted secondary structure of the 264 nt capsid-coding region with an estimated minimum free energy (Delt aG degrees) of -77.16 kcal mol-l. Our chemical probing analysis of the free 264 nt RNA molecule in solution supports the predicted presence of stem-lo ops I, II and III, but casts doubts on stem-loop IV. It suggests, instead, the presence of a stem loop TVA 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 res tored by compensatory sequence changes to regenerate individual stem-loop s tructures. 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 translatio n. Another stem-loop predicted by MFOLD that consists of a major portion of the DB sequence was examined by chemical probing but found little experime ntal support. Changes of the DB sequence without affecting the postulated s tem structure led to drastic losses of translation efficiency. Thus, a simp le structural basis for the enhanced translation could be that stem-loops I , II, III and IVA and the UCUCC sequence may facilitate the interaction bet ween DB and the anti-DB in 16 S-like rRNA in initiating translation of GLV mRNA in G. lamblia. (C) 2001 Academic Press.