Structural determinants of an internal ribosome entry site that direct translational reading frame selection

The dicistrovirus intergenic internal ribosome entry site (IGR IRES) directly recruits the ribosome and initiates translation using a non-AUG codon. A sub-set of IGR IRESs initiates translation in either of two overlapping open reading frames (ORFs), resulting in expression of the 0 frame viral structural polypro-tein and an overlapping +1 frame ORFx. A U–G base pair adjacent to the anticodon-like pseudoknot of the IRES directs +1 frame translation. Here, we show that the U-G base pair is not absolutely required for +1 frame translation. Extensive mutagenesis demon-strates that 0 and +1 frame translation can be uncou-pled. Ribonucleic acid (RNA) structural probing anal-yses reveal that the mutant IRESs adopt distinct con-formations. Toeprinting analysis suggests that the reading frame is selected at a step downstream of ri-bosome assembly. We propose a model whereby the IRES adopts conformations to occlude the 0 frame aminoacyl-tRNA thereby allowing delivery of the +1 frame aminoacyl-tRNA to the A site to initiate trans-lation of ORFx. This study provides a new paradigm for programmed recoding mechanisms that increase the coding capacity of a viral genome