Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association

Sprink, Thiemo; Ramrath, D.J.F.; Yamamoto, Hiroshi; Yamamoto, Kaori; Loerke, Justus; Ismer, Jochen; Hildebrand, Peter W.; Scheerer, Patrick; Bürger, Jörg; Mielke, Thorsten; Spahn, C.M.T.

doi:10.1126/sciadv.1501502

Cited by 66 publications

(124 citation statements)

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“…1 a,b). Despite the fact that the tRNA is not in the P/I state interacting via its CCA end with eIF5B, as it would be if it were still acylated, the degree of relative rotation of the subunits and the overall conformation of eIF5B, is in agreement with both our previous lower resolution reconstruction [9] as well as with the recent reconstruction of the bacterial counterpart initiation complex with IF2 [14]. We therefore conclude that the conformation observed here for eIF5B represents that during initiation just before GTP hydrolysis.…”

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confidence: 78%

“…A similar configuration has been recently observed in a high resolution cryoEM study for the bacterial complex with fMet-tRNA fMet , IF2 and a non-hydrolysable GTP analog [14]. Two mayor populations could be resolved in the bacterial dataset, corresponding to two different rotational sates of the small ribosomal subunit (30S).Thus, in the most rotated structure of the bacterial complex (termed ICI), the bacterial equivalent of Tyr837 is modeled in a position not aligned with the catalytic histidine (Fig.…”

mentioning

confidence: 60%

“…2c, second panel from the left) and the histidine itself is not in the fully activated conformation seen here. The second structure present in the bacterial complex (termed ICII), exhibits an intermediate degree of small subunit rotation similar to the one described here, but unfortunately, the limited local resolution of this complex in that area meant that there was no description of the particular tyrosine [14]. Given the conservation of the factor and the tyrosine in particular (Fig.…”

Section: Cc-by-nc-nd 40 International License Not Peer-reviewed) Is mentioning

confidence: 99%

“…(c) Diagram of sequence of events described in (a) showing the region around the γ-phosphate of GTP (blue). Adapted from PDB-IDs 4NCN, 4NCF [7] and 3JCJ, 3JCN [14]. This sequence of events is suggested by a common activation mechanism for translational GTPases implied by structural data.…”

Section: Figurementioning

confidence: 99%

“…Starting from the left: interchange of GDP (pink) to GTP (orange) allows for the recognition of the initiator aminoacyl-tRNA (green) by eIF5B (red) in the context of the smal ribosomal subunit (40S, yellow). Recruitment of the large ribosomal subunit (60S, blue) is facilitated by eIF5B-GTP in the fully rotated state of the 40S [14]. Partial back-rotation of the 40S results in interaction of a universally conserved tyrosine and the active site histidine, which results in a catalytically active form for GTP hydrolysis.…”

Section: Figurementioning

confidence: 99%

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GTP Hydrolysis by eIF5B in the Last Step of Translation Initiation Is Activated by a Rotation of the Small Ribosomal Subunit

Fernandez

Ramakrishnan

2017

Preprint

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Placement of an initiator aminoacyl-tRNA [(f)Met-tRNAi (f)Met ] base paired with the AUG initiation codon of a messenger RNA (mRNA) is the first step of translation. The eukaryotic translation factor eIF5B or its bacerial homologue IF2 facilitate the correct positioning of initiator tRNA in the P site of the ribosome. We report the electron cryomicroscopy (cryoEM) structure of a stabilized intermediate state of a yeast 80S/tRNAiMet /eIF5B complex at 3.6Å resolution. The structure shows how a universally conserved tyrosine couples the rotational state of the small ribosomal subunit with GTP hydrolysis. * To whom correspondence should be addressed at isf2106@cumc.columbia.edu and ramak@mrc-lmb.cam.ac.uk 1 . CC-BY-NC-ND 4.0 International license not peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was . http://dx.doi.org/10.1101/172825 doi: bioRxiv preprint first posted online Aug. 4, 2017; Initiator aminoacyl-tRNA (fMet-tRNA i fMet in Bacteria, Met-tRNA i Met in Archaea and Eukarya) is the only aminoacyl-tRNA delivered to the peptidyl site (P site) of the ribosome during the initiation stage of translation [1]. Since initiation is not only a rate-limiting step but also of fundamental importance in setting up the correct reading frame, the molecular mechanisms involved in this step are complex and tightly regulated.The detailed process varies significantly in eukaryotes, where the multisubunit factor eIF4F binds to the 5' cap of mRNA and recruites the 43S complex of the small subunit (40S) with factors eIF3, eIF2, eIF1, eIF1A, and eIF5 [2,3].This 48S complex scans along the mRNA until the start codon is reached. This results in conformational changes that lead to GTP hydrolysis by eIF2 and dissociation of the various initiation factors [4]. In the final step, binding of the GTPase eIF5B results in recruitment of the large subunit (60S), followed by GTP hydrolysis and dissociation of eIF5B [1].Just three of the eukaryotic initiation factors have orthologs in bacteria, and recent work suggests that certain aspects of the mechanism involving these three factors are conserved in both domains of life [5].In particular, the final step involving recruitment of the large subunit by IF2, the bacterial counterpart of eIF5B, is probably the most similar. However, the considerable biochemical and structural data for the bacterial case contrasts with the relatively poor information for the eukaryotic case.Structures of both archaeal aIF2 [6] and eukaryotic eIF5B [7]have been solved in isolation. Recent studies on yeast eIF5B propose a "domain release" mechanism by which domains III and IV of eIF5B (and the α-helix connecting them, h12) change conformation dramatically with respect to the G domain and domain II upon GTP binding [7]. These conformational changes in domains III and IV are important for Met-tRNA i Met binding (domain IV [8]) and ribosome interaction (domain III [9]).We previously reported a cryoEM structure at 6.6Å resolution of a yeast initia...

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confidence: 78%

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confidence: 60%

Section: Cc-by-nc-nd 40 International License Not Peer-reviewed) Is mentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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