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. 2020 Apr 14;9:e54575. doi: 10.7554/eLife.54575

Figure 6. The 5'-UTR-IRES requires TC, eIF1 or eIF1A to assemble a functional initiation complex via an uAUG intermediate.

(A) Toe-print analysis of 48S initiation complexes assembled on 5'-UTR-IRES in an in vitro reconstituted system. eIF2 delivers Met-tRNAiMet to the uAUG (lane 2) and requires the presence of eIF1 or eIF1A to transition to the bona fide AUG codon of ORF1 (lane 3). Under some conditions, eIF5B can substitute for eIF2 in Met-tRNAiMet delivery (Terenin et al., 2008). In the absence of eIF1 or eIF1A, a robust toe-print signal is detected in the presence of eIF5B (lane 4), however, Met-tRNAiMet is delivered to the uAUG, and thus eIF5B is unable to find the annotated AUG even in the presence of eIF1 or eIF1A (lane 5). (B) A model for 5'-UTR-IRES-mediated translation initiation. From the bottom right: injection of the genomic (+)-ssRNA of the CrPV into the cytosol allows the 5'-UTR-IRES to capture free 40S and eIF3, which is recruited to the complex with an initial canonical conformation of the 40S head without tilt or swivel. Insertion of 5'-UTR-IRES DIII in the vicinity of the E-site and a swiveling movement of the 40S head induces a ‘locked’ conformation of the complex, with the uAUG at 701 in the vicinity of the P-site. Delivery of Met-tRNAiMet and location of ORF1 AUG is achieved by the concerted action of eIF2, eIF1 and eIF1A. Large subunit recruitment mediated by eIF5B will allow transitioning into elongation.

Figure 6.

Figure 6—figure supplement 1. Control toe-print experiments.

Figure 6—figure supplement 1.

(A) Toe-print analysis for 5'-UTR-IRES, 5'-UTR-IRES–eIF3 and 40S/5'-UTR-IRES–eIF3 complexes (lanes 1, 2 and 3, respectively). No toe-print signal was detected that is ascribable to uAUG or annotated AUG in any of these reactions. Robust toe-print signal could be detected 15–17 nucleotides downstream of uAUG and AUG only for 40S–5'-UTR-IRES–eIF3, and in the presence of TC (eIF2–Met-tRNAiMet–GTP) and eIF1 or eIF1A. (B) The uAUG toe-print signal can be abolished by mutation of the uAUG to ACG, redirecting the Met-tRNAiMet loading event exclusively to the annotated AUG in the presence of TC and eIF1 or eIF1A.