Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition
- MRC Laboratory of Molecular Biology, United Kingdom
- Indian Institute of Science, India
- Shoolini University of Biotechnology and Management Sciences, India
- 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
-
Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map A)Electron Microscopy Data Bank, 4330.
-
Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Sample Map 1)Electron Microscopy Data Bank, 4328.
-
Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map B)Electron Microscopy Data Bank, 4331.
-
Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map C1)Electron Microscopy Data Bank, 4327.
-
Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map C2)Electron Microscopy Data Bank, 4329.
Article and author information
Author details
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.
Reviewing Editor
- Rachel Green, Johns Hopkins School of Medicine, United States
Version history
- Received: June 26, 2018
- Accepted: November 21, 2018
- Accepted Manuscript published: November 26, 2018 (version 1)
- Accepted Manuscript updated: November 29, 2018 (version 2)
- Accepted Manuscript updated: November 30, 2018 (version 3)
- Version of Record published: December 18, 2018 (version 4)
- Version of Record updated: January 10, 2019 (version 5)
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
-
- 4,310
- views
-
- 710
- downloads
-
- 77
- 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)
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
Further reading
-
- Structural Biology and Molecular Biophysics
Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson’s disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Several structures of LRRK2 have been reported, but structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single-particle cryo-electron microscopy to solve the structure of a bacterial Roco protein (CtRoco) in its GTP-bound state, aided by two conformation-specific nanobodies: NbRoco1 and NbRoco2. This structure presents CtRoco in an active monomeric state, featuring a very large GTP-induced conformational change using the LRR-Roc linker as a hinge. Furthermore, this structure shows how NbRoco1 and NbRoco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights into the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity.
-
- Developmental Biology
- Structural Biology and Molecular Biophysics
A crucial event in sexual reproduction is when haploid sperm and egg fuse to form a new diploid organism at fertilization. In mammals, direct interaction between egg JUNO and sperm IZUMO1 mediates gamete membrane adhesion, yet their role in fusion remains enigmatic. We used AlphaFold to predict the structure of other extracellular proteins essential for fertilization to determine if they could form a complex that may mediate fusion. We first identified TMEM81, whose gene is expressed by mouse and human spermatids, as a protein having structural homologies with both IZUMO1 and another sperm molecule essential for gamete fusion, SPACA6. Using a set of proteins known to be important for fertilization and TMEM81, we then systematically searched for predicted binary interactions using an unguided approach and identified a pentameric complex involving sperm IZUMO1, SPACA6, TMEM81 and egg JUNO, CD9. This complex is structurally consistent with both the expected topology on opposing gamete membranes and the location of predicted N-glycans not modeled by AlphaFold-Multimer, suggesting that its components could organize into a synapse-like assembly at the point of fusion. Finally, the structural modeling approach described here could be more generally useful to gain insights into transient protein complexes difficult to detect experimentally.
