Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition

Abstract
Data availability
Article and author information
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Abstract

In eukaryotic translation initiation AUG recognition of the mRNA requires accommodation of Met-tRNA_i in a 'P_IN' 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-tRNA_i. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNA_i interaction influenced initiation at near-cognate UUG codons in 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.

Data availability

Five maps have been deposited in the EMDB with accession codes EMDB: 4328, EMDB: 4330, EMDB: 4331, EMDB: 4327, EMDB: 4329, for the sample 1 map, Map A, Map B, Map C1 and Map C2, respectively. Two atomic coordinate models have been deposited in the PDB with accession codes PDB: 6FYX, PDB: 6FYY, for models showing TC in conformation 1 and conformation 2, respectively.All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Tables 4 and 5 and Figures 4 and 5

The following data sets were generated

(2018) Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map A)
Electron Microscopy Data Bank, 4330.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4330
(2018) Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Sample Map 1)
Electron Microscopy Data Bank, 4328.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4328
(2018) Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map B)
Electron Microscopy Data Bank, 4331.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4331
(2018) Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map C1)
Electron Microscopy Data Bank, 4327.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4327
(2018) Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map C2)
Electron Microscopy Data Bank, 4329.

https://www.ebi.ac.uk/pdbe/entry/emdb/EMD-4329
(2018) Date from: Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition
Protein Data Bank, 6FYX.

http://www.rcsb.org/structure/6FYX
(2018) Date from: Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition
Protein Data Bank, 6FYY.

http://www.rcsb.org/structure/6FYY

Article and author information

Author details

José Luis Llácer

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0001-5304-1795
Tanweer Hussain

Molecular Reproduction, Development and Genetics (MRDG), Indian Institute of Science, Bangalore, India

Competing interests
No competing interests declared.
Adesh K Saini

Shoolini University of Biotechnology and Management Sciences, Solan, India

Competing interests
No competing interests declared.
Jagpreet Singh Nanda

Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

Competing interests
No competing interests declared.
Sukhvir Kaur

Shoolini University of Biotechnology and Management Sciences, Solan, India

Competing interests
No competing interests declared.
Yuliya Gordiyenko

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

Competing interests
No competing interests declared.
Rakesh Kumar

Shoolini University of Biotechnology and Management Sciences, Solan, India

Competing interests
No competing interests declared.
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

For correspondence
alanh@mail.nih.gov

Competing interests
Alan G Hinnebusch, Reviewing editor, eLife.
Jon R Lorsch

Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States

For correspondence
jon.lorsch@nih.gov

Competing interests
No competing interests declared.

"This ORCID iD identifies the author of this article:" 0000-0002-4521-4999
V Ramakrishnan

MRC Laboratory of Molecular Biology, Cambridge, United Kingdom

For correspondence
ramak@mrc-lmb.cam.ac.uk

Competing interests
No competing interests declared.

Funding

Medical Research Council (MC_U105184332)

V Ramakrishnan

Wellcome (WT096570)

V Ramakrishnan

Agouron Institute

V Ramakrishnan

Department of Science and Technology, Ministry of Science and Technology (Int/NZ/P-2/13)

Adesh K Saini

National Institutes of Health (GM62128)

Jon R Lorsch

Human Frontier Science Program (RGP-0028/2009)

Alan G Hinnebusch

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.