eIF2α interactions with mRNA control accurate start codon selection by the translation preinitiation complex

Accurate identification of the translation initiation codon is critical to ensure the synthesis of the correct cellular proteins in the proper amounts. In eukaryotes, translation initiation generally occurs by a cap-dependent scanning mechanism, wherein the small (40S) subunit of the ribosome recruits methionyl initiator tRNA (Met-tRNAi) in a ternary complex (TC) with GTP-bound eukaryotic initiation factor 2 (eIF2), in a reaction stimulated by factors eIF1, eIF1A, and eIF3. The resulting 43S preinitiation complex (PIC) attaches to the capped 5’ end of mRNA and scans the mRNA leader for an AUG start codon. The nucleotide sequence immediately surrounding the start codon—the “Kozak context”—particularly at the -3 and +4 positions (numbered from the A of AUG (+1)) influences the efficiency of start codon selection. AUG recognition triggers rearrangement of the 48S preinitiation complex (PIC) from an open conformation to a closed state with more tightly-bound Met-tRNAi. Cryo-EM structures have revealed interactions unique to the closed complex between arginines R55/R57 of eIF2α with mRNA, including the -3 nucleotide of the “Kozak’’ context (Fig A).

In recent work of our lab with collaboration of Dr. Alan Hinnebusch’s Lab (NIH,USA), we found that eIF2α-R55/R57 substitutions reduced recognition of a UUG start codon at HIS4 mRNA in vitro, R55G/R57E accelerated dissociation of the eIF2·GTP·Met-tRNAi ternary complex (TC) from reconstituted PICs with a UUG start codon, indicating destabilization of the closed complex. R55/R57 substitutions also decreased usage of poor-context AUGs in SUI1 and GCN4 mRNAs in vivo. In contrast, eIF2α-R53 interacts with the rRNA backbone only in the open complex (Fig B), and the R53E substitution enhanced initiation at a UUG codon and poor-context AUGs, while reducing the rate of TC loading in vivo. Consistently, R53E slowed TC binding to the PIC while decreasing TC dissociation at UUG codons in vitro, indicating destabilization of the open complex. Thus, distinct interactions of eIF2α with rRNA or mRNA stabilize first the open, and then closed, conformation of the PIC to influence the accuracy of initiation in vivo.

For full article: https://academic.oup.com/nar/article/48/18/10280/5909532