Identification of an essential pseudoknot in the putative downstream internal ribosome entry site in giardiavirus transcript

Enhanced translation of giardiavirus-luciferase chimeric mRNA in Giardia lamblia requires the initial 264-nt viral capsid coding region as a putative internal ribosomal entry site (IRES). Essential structural elements in this site include (1) a downstream box (1313) complementary to the anti-DB at the 3' end of 16S-like rRNA, (2) stem-loops 1, 11, III, and IVA, and (3) a pentanucleotide 5'-UCUCC-3' immediately downstream from stem loop IVA. A search for the structural role of the pentanucleotide suggested that it may form a pseudoknot with another pentanucleotide 5'-GGAGA-3' in loop Ill. Alteration of the two pentanucleotides by site-directed mutagenesis resulted in a drastic reduction in translation of the transcript. But the loss was recovered by compensatory changes in the two sequences, suggesting Watson-Crick base pairings between them. Results from in vitro enzymatic and chemical structural probing supported the presence of such a pseudoknot 143 nt downstream from the initiation codon. Minor repositioning of this codon led invariably to a complete loss of translation, suggesting that the initiation site is confined within a rigid position defined by all the structural elements in the IRES including the pseudoknot. This is the first pseudoknot of its kind shown to play an important role in a downstream IRES of a viral transcript. The finding is particularly interesting because it could reflect a unique feature of translation initiation in Giardia, which is known to have exceedingly short (1-6 nt) 5' untranslated regions in its mRNAs.