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

Capped mRNA with a Single Nucleotide Leader Is Optimally Translated in a Primitive Eukaryote, Giardia lamblia

“…5C), known to hydrolyze only RNA structures in stacked helical conformations (36). The results indicate that the enzyme-digested RNA molecule provided reverse transcriptase stops at positions G1, A2, G4, U42, C43, U44, U46, and C47 in the postulated stem 1 structure and at positions C10, G11, C12, C13, G34, and G37 in the postulated stem II (Fig.…”

“…This downstream box is not involved in any secondary structure of the viral transcript (35) but is essential for initiating translation of the viral transcript (12), suggesting that a Shine-Dalgarno type interaction could be involved in translation initiation, albeit by a sequence downstream from the initiation codon. Further analysis indicated that translation of the uncapped giardiaviral transcript in Giardia is initiated at an internal ribosomal entry site in the mRNA covering parts of both the 5Ј-UTR and the downstream region including the downstream box (36). Within the complex secondary structures of this internal ribosomal entry site, there is a stem-loop structure located 21 nucleotides upstream from the AUG and another stem-loop 7 nucleotides downstream from it, both playing essential roles in translation initiation.…”

“…Within the complex secondary structures of this internal ribosomal entry site, there is a stem-loop structure located 21 nucleotides upstream from the AUG and another stem-loop 7 nucleotides downstream from it, both playing essential roles in translation initiation. The very location of the AUG between the two stem-loops cannot be altered; a mere shift upstream or downstream by three nucleotides completely abrogated translation initiation (36). Thus, the 31-nucleotide stretch between the two stem-loops, which could accommodate the size of a bound small ribosome subunit well (37,38), may be the very site for recruited ribosome small subunit to bind where the initiation codon in the mRNA may fit into the precise site of the bound ribosome for initiating the translation.…”

“…Enzymatic probing of the RNA structure was carried out essentially as described previously (36). The capped in vitro transcript of GGAUGFluc (2 g), together with 4 g of yeast tRNA, was denatured in the 1ϫ RNA structure buffer (Ambion) at 65°C for 15 min and then cooled slowly to ambient temperature within 30 min.…”

Structural Elements in the 5′-Untranslated Region of Giardiavirus Transcript Essential for Internal Ribosome Entry Site-Mediated Translation Initiation

“…Functional and structural analysis of the 264-nt coding region of the IRES were conducted, and several structural elements essential for translation initiation were identified (4). They include a 13-nt downstream box (DB) at positions 66 to 78 that complements (with two gaps) a 15-nt sequence near the 3Ј end of Giardia 16S-like rRNA (29), stem-loops I (nt 11 to 35), II (nt 144 to 164), III (nt 166 to 182), and IVA (nt 193 to 215), and a novel pseudoknot structure between loop II and a region downstream from stem-loop IVA (4,5). Mutations that destroyed the stems in stem-loops I and II or disrupted the pairing of DB with rRNA resulted in a drastic reduction in IRES-mediated translation.…”