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

  1. José Luis Llácer
  2. Tanweer Hussain
  3. Adesh K Saini
  4. Jagpreet Dabas Nanda
  5. Sukhvir Kaur
  6. Yuliya Gordiyenko
  7. Rakesh Kumar
  8. Alan G Hinnebusch  Is a corresponding author
  9. Jon R Lorsch  Is a corresponding author
  10. Venki Ramakrishnan  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom
  2. Indian Institute of Science, India
  3. Shoolini University of Biotechnology and Management Sciences, India
  4. National Institutes of Health, United States

Abstract

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 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

Article and author information

Author details

  1. José Luis Llácer

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5304-1795
  2. Tanweer Hussain

    Molecular Reproduction, Development and Genetics (MRDG), Indian Institute of Science, Bangalore, India
    Competing interests
    No competing interests declared.
  3. Adesh K Saini

    Shoolini University of Biotechnology and Management Sciences, Solan, India
    Competing interests
    No competing interests declared.
  4. Jagpreet Dabas 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.
  5. Sukhvir Kaur

    Shoolini University of Biotechnology and Management Sciences, Solan, India
    Competing interests
    No competing interests declared.
  6. Yuliya Gordiyenko

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    No competing interests declared.
  7. Rakesh Kumar

    Shoolini University of Biotechnology and Management Sciences, Solan, India
    Competing interests
    No competing interests declared.
  8. 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.
  9. 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.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4521-4999
  10. Venki 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)

  • Venki Ramakrishnan

Wellcome (WT096570)

  • Venki Ramakrishnan

Agouron Institute

  • Venki 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.

Metrics

  • 4,704
    views
  • 745
    downloads
  • 84
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. José Luis Llácer
  2. Tanweer Hussain
  3. Adesh K Saini
  4. Jagpreet Dabas Nanda
  5. Sukhvir Kaur
  6. Yuliya Gordiyenko
  7. Rakesh Kumar
  8. Alan G Hinnebusch
  9. Jon R Lorsch
  10. Venki Ramakrishnan
(2018)
Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition
eLife 7:e39273.
https://doi.org/10.7554/eLife.39273

Share this article

https://doi.org/10.7554/eLife.39273

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Jie Luo, Jeff Ranish
    Tools and Resources

    Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.

    1. Structural Biology and Molecular Biophysics
    Yuanyuan Wang, Fan Xu ... Yongning He
    Research Article

    SCARF1 (scavenger receptor class F member 1, SREC-1 or SR-F1) is a type I transmembrane protein that recognizes multiple endogenous and exogenous ligands such as modified low-density lipoproteins (LDLs) and is important for maintaining homeostasis and immunity. But the structural information and the mechanisms of ligand recognition of SCARF1 are largely unavailable. Here, we solve the crystal structures of the N-terminal fragments of human SCARF1, which show that SCARF1 forms homodimers and its epidermal growth factor (EGF)-like domains adopt a long-curved conformation. Then, we examine the interactions of SCARF1 with lipoproteins and are able to identify a region on SCARF1 for recognizing modified LDLs. The mutagenesis data show that the positively charged residues in the region are crucial for the interaction of SCARF1 with modified LDLs, which is confirmed by making chimeric molecules of SCARF1 and SCARF2. In addition, teichoic acids, a cell wall polymer expressed on the surface of gram-positive bacteria, are able to inhibit the interactions of modified LDLs with SCARF1, suggesting the ligand binding sites of SCARF1 might be shared for some of its scavenging targets. Overall, these results provide mechanistic insights into SCARF1 and its interactions with the ligands, which are important for understanding its physiological roles in homeostasis and the related diseases.