Structural basis for non-AUG translation regulation by 5MPs.

Area of Science:

• Molecular Biology
• Structural Biology
• Genetics

Background:

• Cellular proteome regulation involves translation initiation at AUG or non-canonical (non-AUG) start codons.
• Non-AUG initiation is crucial during stress and linked to diseases like cancer.
• eIF5-mimic proteins (5MPs) limit non-AUG start codon usage, reprogramming proteoform expression from mRNAs with alternative start sites.

Purpose of the Study:

• To elucidate the mechanism by which 5MPs induce translational reprogramming.
• To understand how 5MPs restrict non-AUG start codon usage.

Main Methods:

• In extracto cryo-electron microscopy (cryo-EM)
• Biochemical assays
• Analysis of 5MP-bound 48S pre-initiation complexes (PICs) from native cell extracts.

Main Results:

• Translational repression by 5MP is sequence context-dependent near start codons.
• Cryo-EM structures show 5MP binding at the A site of the small ribosomal subunit, stabilizing an open-head PIC conformation.
• 5MP's N-terminal region blocks the A site, while the C-terminal domain interacts with eIF2β and initiator tRNA outside the P site (Pout).

Conclusions:

• 5MP directly biases initiating 48S complexes toward an open conformation.
• This bias promotes mRNA scanning and inhibits initiation at suboptimal start codons.
• The findings provide a structural mechanism for 5MP-mediated translational control.