Global analysis of translation termination in E. coli

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• Accurate protein translation termination is crucial for protein fidelity and ribosome recycling.
• Bacterial release factors (RFs) mediate stop codon recognition and termination; RF3 aids RF dissociation.
• Previous studies used reporter constructs to examine RF mutations but lacked a genome-wide perspective.

Purpose of the Study:

• To globally analyze the impact of release factor mutations on translation using ribosome profiling.
• To investigate genome-wide effects of impaired translation termination on ribosome occupancy and gene expression.

Main Methods:

• Ribosome profiling was performed on isogenic bacterial strains with characterized release factor mutations.
• Analysis focused on ribosome accumulation over stop codons and in regions downstream of stop codons.
• Specific cases of readthrough and frameshifting were confirmed.

Main Results:

• Increasing severity of release factor defects correlated with increased ribosome accumulation over stop codons.
• A significant number of genes (≥ 50) showed ribosome signatures suggesting translation past the stop codon.
• RF3 deletion exacerbated termination defects; native E. coli K-12 showed readthrough signatures, especially at UGA codons.

Conclusions:

• Translation termination is generally efficient but imperfect, with readthrough and frameshifting occurring at specific genes.
• Release factor mutations enhance recoding events and disrupt operon attenuation control.
• These alterations can expand the protein repertoire or produce deleterious proteins, impacting cellular function.