Molecular architecture of a eukaryotic translational initiation complex

By I. S. Fernández, X. Bai, T. Hussain, A. Kelley, J. Lorsch, V. Ramakrishnan, Sjors H. W. Scheres. 2013

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Introduction Initiation of protein synthesis is a key step in the control of gene expression. In eukaryotes, initiation is a highly complex process that requires almost a dozen protein factors. The last step involves joining of the large and small subunits of the ribosome to form the 80S initiation complex with the transfer RNA (tRNA) in the P-site base paired to the start codon. This step is catalyzed by the guanosine triphosphatase (GTPase) factor eIF5B. In addition, eIF5B is thought to play a role in ensuring that translation initiation takes place only on mature ribosomes. The complex of the ribosome with eIF5B and initiator tRNA determined by cryo-EM, showing conformational changes in all three components. (A) The structure of the initiation complex of the ribosome with initiation factor eIF5B, initiator tRNA, and mRNA start codon. (B) Comparison with the canonical ribosome (gray) reveals a rotation of the two subunits relative to each other. (C) There are large conformational changes in eIF5B and initiator tRNA relative to the isolated structures. Methods The development of fast, direct electron detectors and new methods of image analysis for cryo–electron microscopy allow high-resolution reconstructions from much smaller numbers of particles than previously possible. We used these new methods to provide feedback to improve the biochemical preparation of samples for structure determination of the eukaryotic translation initiation complex with initiator tRNA and eIF5B, which was trapped on the ribosome with the nonhydrolyzable GTP analog GDPCP. Results Although the structure of the fully assembled complex was calculated from only 5143 particles, representing just 3% of the population in the sample, it was possible to obtain a resolution of 6.6 Å. This allowed us to propose a molecular model for the initiation complex. The structure shows that the subunits of the ribosome are rotated relative to the canonical state after initiation. The long helix and C-terminal domain of eIF5B have changed conformation and moved into the ribosome, where the C-terminal domain interacts with the initiator tRNA. The tRNA is stabilized in a distorted conformation, with its 3′-CCA end out of the peptidyl transferase center. Discussion The conformational change in eIF5B may be induced upon binding to the small (40S) subunit when it specifically recognizes initiator tRNA. In its altered conformation, eIF5B interacts simultaneously with the initiator tRNA and the GTPase center of the ribosome, thus coupling GTP hydrolysis with tRNA recognition in the ribosome. The large number of contacts made by eIF5B with the ribosomal subunits and tRNA is consistent with its role in subunit joining. A close contact with the eukaryote-specific ribosomal protein L40 would not be possible in the immature ubiquitinated form of L40, thus precluding the recruitment of immature 60S subunits. A comparison with previous work on the bacterial homolog IF2 suggests that the mechanism of this step of initiation is conserved across kingdoms. Finally, the use of recent advances in cryo-EM to determine a relatively high-resolution structure of the eIF5B-ribosome complex from a very small fraction of a sample could be a general approach for the study of other dynamic or transient biological complexes. Dissecting Initiation Ribosomes make proteins. The initiation step of protein synthesis involves initiation factors (IFs) that help to position the initiation codon of the messenger RNA (mRNA) and the initiator transfer RNA. The initiation factor eIF5B is universally conserved, and promotes recruitment of the large ribosomal subunit during the terminal step of initiation. Fernández et al. (p. 10.1126/science.1240585, published online 8 November) used cryoelectron microscopy to determine the structure of the yeast eIF5B initiation complex and provide a snapshot of the final step of eukaryotic translation initiation. Cryo–electron microscopy allows the rapid analysis of a yeast ribosome-initiator complex from a tiny data set. The last step in eukaryotic translational initiation involves the joining of the large and small subunits of the ribosome, with initiator transfer RNA (Met-tRNAiMet) positioned over the start codon of messenger RNA in the P site. This step is catalyzed by initiation factor eIF5B. We used recent advances in cryo–electron microscopy (cryo-EM) to determine a structure of the eIF5B initiation complex to 6.6 angstrom resolution from <3% of the population, comprising just 5143 particles. The structure reveals conformational changes in eIF5B, initiator tRNA, and the ribosome that provide insights into the role of eIF5B in translational initiation. The relatively high resolution obtained from such a small fraction of a heterogeneous sample suggests a general approach for characterizing the structure of other dynamic or transient biological complexes.

Subfields: Molecular Biology

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Citations in the corpus

The Jigsaw Puzzle of mRNA Translation Initiation in Eukaryotes: A Decade of Structures Unraveling the Mechanics of the Process (2018, background): “It was only after the determination of the structure of 80S-Met-tRNAi-mRNA-eIF5B that the molecular mechanics of eIF5B action were unraveled (37).”
Initiation factor 2 stabilizes the ribosome in a semirotated conformation (2015, background): “4° rotation of the small ribosomal subunit relative to the large ribosomal subunit (41).”
Structural Insights into the Mammalian Late-Stage Initiation Complexes (2020, background): “In the fourth and final step, ABCE1 is replaced by the GTPase eIF5B on helix 14 of 18S rRNA, thus stimulating the joining of the 40S and 60S ribosomal subunits, forming an 80S complex (Fernandez et al., 2013), and eIF1A and eIF5B are released together (Fringer et al.”
Protein Synthesis Initiation in Eukaryotic Cells. (2018, background): “There is a large change in the conformation of 80S-bound eIF5B, where domains III and IV are rotated 60°–65° relative to free eIF5B (Fernandez et al. 2013; Yamamoto et al. 2014).”
eIF5B employs a novel domain release mechanism to catalyze ribosomal subunit joining (2014, methodology): “Under the assumption that the interactions between domain II and the 18S rRNA are the last to be broken (as they do not directly depend on the presence of GTP as do those of the G domain with the SRL), domain III moves away from the SRL and domain IV moves away from the peptidyl-transferase center (”
Long-range interdomain communications in eIF5B regulate GTP hydrolysis and translation initiation (2020, methodology): “Much is known about the ribosome-binding determinants of eIF5B (21) as well as how the Met-tRNAi Met is recognized (22).”
Single-Particle Cryo-EM at Crystallographic Resolution. (2015, background): “…a number of conformations of the molecule being studied, or separate fully intact particles or complexes from incomplete, truncated or fragmented complexes, or from those damaged during vitrification (Fernández et al., 2013; 454 Cell 161, April 23, 2015 ª2015 Elsevier Inc. Liao et al., 2014).”