Science2013-11-16 11:47 PM

Molecular Architecture of a Eukaryotic Translational Initiation Complex

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.
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.
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.
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.






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