Abstract
Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a "dead-end" intermediate at 3.6 Å allowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states.
Highlights
Translation in higher eukaryotes is a complex endeavor [1]
Once a full (80S) ribosome is assembled with a proper AUG codon at the P site that is base-paired with Met-tRNAiMet, the ribosome is competent for elongation [16]
The first eIF5B-ribosome association occurs in the context of the 40S: immediately after eIF2/eIF1 are released from the 48S complex, eIF5B interacts via its domain II with the 40S, and the flexible unit formed by domains III and IV “senses” the presence of Met-tRNAiMet at the P site [23]
Summary
Translation in higher eukaryotes is a complex endeavor [1]. Eukaryotic ribosomes have to synthetize proteins, but they are required to do so in a strictly regulated fashion by delivering proteins in specific cellular contexts in space and time [2]. The bulk of eukaryotic translation regulation is implemented at the first phase of translation, known as initiation [8] In eukaryotes, this phase is very sophisticated and is initiated by the establishment of the 43S Preinitiation Complex (43S-PIC), where the small (40S) ribosomal subunit recruits initiation factors eIF1/1A/3 and 5 and a specific initiator aminoacyl-tRNA (Met-tRNAiMet) in the form of a ternary complex (TC) with eIF2 and GTP [9]. A second GTP-regulated step is required for the recruitment of the large (60S) ribosomal subunit [14] This is catalyzed by another initiation factor, eIF5B [15]. If domain IV is stabilized due to proper recognition of the initiator aminoacyl-tRNA, 60S recruitment will proceed via the increased 60S/40S-interacting surface due to the presence of eIF5B [15, 21]
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