Interface between 40S exit channel protein uS7/Rps5 and eIF2α modulates start codon recognition in vivo

Area of Science:

• Molecular Biology
• Protein Structure and Dynamics
• Eukaryotic Gene Expression

Background:

• The eukaryotic pre-initiation complex (PIC) is crucial for initiating protein synthesis by scanning mRNA for start codons.
• Accurate recognition of the AUG start codon involves a conformational switch of the PIC from an open to a closed state.
• The role of the interface between ribosomal protein uS7 and translation factor eIF2α in this conformational change was previously unclear.

Purpose of the Study:

• To elucidate the functional significance of the uS7/eIF2α interface remodeling in PIC conformational changes.
• To determine how this interface contributes to the fidelity of start codon selection during translation.
• To investigate the dynamic interplay between PIC states and translation initiation accuracy.

Main Methods:

• Utilized cryo-electron microscopy (cryo-EM) reconstructions of yeast PICs to analyze structural changes.
• Employed site-directed mutagenesis of the uS7 protein to disrupt specific interactions with eIF2α.
• Assessed the impact of uS7 mutations on ternary complex (TC) binding and initiation at various start codons in reconstituted PICs.

Main Results:

• uS7 substitutions disrupting eIF2α contacts in the open PIC state led to increased initiation at non-AUG sites.
• A specific uS7 mutation (uS7-S223D) inappropriately stabilized TC binding, suggesting a failure to transition to the closed state.
• Mutations affecting the closed-state uS7-eIF2α interaction resulted in hyperaccurate start codon selection and altered TC dissociation rates.

Conclusions:

• Remodeling of the uS7/eIF2α interface is essential for stabilizing the open PIC conformation, facilitating scanning.
• This interface remodeling further stabilizes the closed PIC conformation, ensuring accurate AUG selection.
• The dynamic regulation of the uS7/eIF2α interface is critical for fidelity in eukaryotic translation initiation.