Specific functional interactions of nucleotides at key -3 and +4 positions flanking the initiation codon with components of the mammalian 48S translation initiation complex

Abstract

Eukaryotic initiation factor (eIF) 1 maintains the fidelity of initiation codon selection and enables mammalian 43S preinitiation complexes to discriminate against AUG codons with a context that deviates from the optimum sequence GCC(A/G)CCAUGG, in which the purines at ^-3 and ⁺4 positions are most important. We hypothesize that eIF1 acts by antagonizing conformational changes that occur in ribosomal complexes upon codon-anticodon base-pairing during 48S initiation complex formation, and that the role of ^-3 and ⁺4 context nucleotides is to stabilize these changes by interacting with components of this complex. Here we report that U and G at ⁺4 both UV-cross-linked to ribosomal protein (rp) S15 in 48S complexes. However, whereas U cross-linked strongly to C₁₆₉₆ and less well to AA_1818-1819 in helix 44 of 18S rRNA, G cross-linked exclusively to AA_1818-1819. U at ^-3 cross-linked to rpS5 and eIF2α, whereas G cross-linked only to eIF2α. Results of UV cross-linking experiments and of assays of 48S complex formation done using α-subunit-deficient eIF2 indicate that eIF2α's interaction with the ^-3 purine is responsible for recognition of the ^-3 context position by 43S complexes and suggest that the ⁺4 purine/AA_1818-1819 interaction might be responsible for recognizing the ⁺4 position.