Long-range interdomain communications in eIF5B regulate GTP hydrolysis and translation initiation

By Bridget Y. Huang, I. S. Fernández. 2020

23 citations recorded by the source corpus.

Significance Translation is a key regulatory step in the control of gene expression. The first stage of translation, initiation, establishes the foundation for the sequential synthesis of a protein. In eukaryotes, 2 GTP-regulated checkpoints ensure the efficiency and fidelity of translation initiation. The GTPase eIF5B is responsible for the correct functioning of the second checkpoint. Molecular interactions of eIF5B with other correctly assembled components on the ribosome lead to GTP hydrolysis that allows the machinery of protein production to transition from initiation into elongation. Here, we show how a highly conserved stretch of residues in eIF5B, identified using electron cryomicroscopy, coordinates the gating into elongation, underscoring the importance of modular architecture in translation factors to sense and communicate ribosomal states. 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.

Subfields: Molecular Biology