Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice K Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States
Sukhvir Kaur
Shoolini University of Biotechnology and Management Sciences, Himachal Pradesh, India
Yuliya Gordiyenko
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
Rakesh Kumar
Shoolini University of Biotechnology and Management Sciences, Himachal Pradesh, India
Alan G Hinnebusch
Laboratory of Gene Regulation and Development, Eunice K Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States
Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice K Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States
V Ramakrishnan
MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNAi in a ‘PIN’ state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNAi. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNAi interaction influenced initiation at near-cognate UUG codonsin vivo, and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.
