Identification of compounds that decrease the fidelity of start codon recognition by the eukaryotic translational...

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Eukaryotic translation initiation involves numerous protein factors.
• Defects in specific factors (e.g., eukaryotic initiation factor 1) can impair start codon fidelity, leading to the Sui(-) phenotype.
• Small molecules that modulate start codon recognition fidelity are currently unknown.

Purpose of the Study:

• To identify small molecules that decrease the fidelity of start codon recognition during translation initiation.
• To develop chemical tools for investigating the molecular mechanisms of translation initiation and regulation.

Main Methods:

• High-throughput screening of over 55,000 compounds using a dual luciferase assay in Saccharomyces cerevisiae.
• Validation of compound effects using in vivo assays monitoring translation from non-AUG start codons.
• Assessment of compound interactions with eukaryotic initiation factor 1 (eIF1) in yeast strains.

Main Results:

• Two structurally related compounds were identified that reduce start codon selection fidelity approximately twofold.
• These compounds enhance translation from non-AUG start codons, including a natural upstream open reading frame initiating at UUG.
• The compounds exacerbate the Sui(-) phenotype caused by eIF1 haploinsufficiency or mutation.
• Compound effects are suppressed by eIF1 overexpression, which restores fidelity in other Sui(-) contexts.

Conclusions:

• The identified compounds directly impact the translational machinery, reducing the accuracy of AUG start codon selection.
• These molecules serve as valuable chemical probes for dissecting the complex process of translation initiation.
• The findings offer new avenues for understanding and potentially manipulating gene expression at the translational level.