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
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Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map A)Electron Microscopy Data Bank, 4330.
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Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Sample Map 1)Electron Microscopy Data Bank, 4328.
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Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map B)Electron Microscopy Data Bank, 4331.
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Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map C1)Electron Microscopy Data Bank, 4327.
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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.
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.
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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
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- Structural Biology and Molecular Biophysics
The functional effects of an RNA can arise from complex three-dimensional folds known as tertiary structures. However, predicting the tertiary structure of an RNA and whether an RNA adopts distinct tertiary conformations remains challenging. To address this, we developed BASH MaP, a single-molecule dimethyl sulfate (DMS) footprinting method and DAGGER, a computational pipeline, to identify alternative tertiary structures adopted by different molecules of RNA. BASH MaP utilizes potassium borohydride to reveal the chemical accessibility of the N7 position of guanosine, a key mediator of tertiary structures. We used BASH MaP to identify diverse conformational states and dynamics of RNA G-quadruplexes, an important RNA tertiary motif, in vitro and in cells. BASH MaP and DAGGER analysis of the fluorogenic aptamer Spinach reveals that it adopts alternative tertiary conformations which determine its fluorescence states. BASH MaP thus provides an approach for structural analysis of RNA by revealing previously undetectable tertiary structures.
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Riboswitches represent a class of non-coding RNA that possess the unique ability to specifically bind ligands and, in response, regulate gene expression. A recent report unveiled a type of riboswitch, known as the guanidine-IV riboswitch, which responds to guanidine levels to regulate downstream genetic transcription. However, the precise molecular mechanism through which the riboswitch senses its target ligand and undergoes conformational changes remain elusive. This gap in understanding has impeded the potential applications of this riboswitch. To bridge this knowledge gap, our study investigated the conformational dynamics of the guanidine-IV riboswitch RNA upon ligand binding. We employed single-molecule fluorescence resonance energy transfer (smFRET) to dissect the behaviors of the aptamer, terminator, and full-length riboswitch. Our findings indicated that the aptamer portion exhibited higher sensitivity to guanidine compared to the terminator and full-length constructs. Additionally, we utilized Position-specific Labelling of RNA (PLOR) combined with smFRET to observe, at the single-nucleotide and single-molecule level, the structural transitions experienced by the guanidine-IV riboswitch during transcription. Notably, we discovered that the influence of guanidine on the riboswitch RNA’s conformations was significantly reduced after the transcription of 88 nucleotides. Furthermore, we proposed a folding model for the guanidine-IV riboswitch in the absence and presence of guanidine, thereby providing insights into its ligand-response mechanism.
