Conformational rearrangements upon start codon recognition in human 48S translation initiation complex

Abstract

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2–GTP–Met-tRNAiMet and eIF3. The ‘open’ 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The ‘closed’ form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A

“…The two helices from eIF3h extend toward the solvent site of the 43S, but the role of eIF3h in initiation was not clear. The core of the mammalian eIF3 octameric structural core is formed by a 7-helix bundle consisting of two helices from eIF3h and one helix from -c, -e, -f, -l and -k ( 4, 29, 32, 39, 40 ). Here, we see a direct interaction between eIF3h and eIF4A-NTD (Fig.…”

Role of aIF5B in archaeal translation initiation

“…Therefore, in order to improve the quality of the cryo-EM maps, we crosslinked IC3 using bis(sulfosuccinimidyl)suberate (BS 3 ) at 51°C as previously done for other ICs ( e.g . ( 78 , 79 ), see Materials and Methods). Cross-linking with BS 3 did not change the overall structure, but it greatly improved the quality of the potential map in the aIF5B binding regions.…”

Role of aIF5B in archaeal translation initiation

“…g . (78,79), see Materials and Methods). Cross-linking with BS 3 did not change the overall structure, but it greatly improved the quality of the potential map in the aIF5B binding regions.…”