Structure of a Gcn2 dimer in complex with the large 60S ribosomal subunit

Area of Science:

• Molecular Biology
• Cellular Biology
• Structural Biology

Background:

• The integrated stress response (ISR) is crucial for eukaryotic cells to adapt to environmental stress.
• Stress triggers ribosome collisions, activating Gcn2 kinase, which phosphorylates eIF2α to regulate translation.
• Structural mechanisms of Gcn2 interaction with ribosomes remained elusive.

Purpose of the Study:

• To elucidate the structural basis of Gcn2 interaction with the ribosomal 60S subunit.
• To understand the role of the Gcn2-ribosome complex in the ISR.
• To investigate the functional implications of Gcn2 binding to the 60S subunit.

Main Methods:

• Ex vivo affinity purification of yeast Gcn2 and ribosomal particles.
• Cryoelectron microscopy (cryo-EM) to determine the structure of the Gcn2-60S complex.
• Functional studies to assess Gcn2 binding and activity.

Main Results:

• A cryo-EM structure revealed a yeast Gcn2 dimer bound to the 60S ribosomal subunit.
• Gcn2 dimerizes via histidine tRNA synthetase-like domains, interacting with the stalk-base and sarcin-ricin loop.
• The C-terminal domain of Gcn2 dimerizes and binds the peptidyl-transferase center, occupying tRNA sites.
• Gcn2 binding to the 60S subunit does not require Gcn1/Gcn20 or lead to eIF2α phosphorylation.
• Stress induces a shift of Gcn2 from the 60S subunit to collided ribosomes.

Conclusions:

• The Gcn2-60S complex represents an inactive stand-by state.
• This complex facilitates rapid Gcn2 redistribution to collided ribosomes upon stress.
• The findings provide structural insights into the ISR and stress granule dynamics.