Miscoding-induced stalling of substrate translocation on the bacterial ribosome

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Ribosome translocation along mRNA dictates protein synthesis speed and accuracy.
• Previous studies suggested late intermediates in translocation involving peptidyl-tRNA P-site entry.
• The role of decoding fidelity in translocation kinetics remained unclear.

Purpose of the Study:

• To investigate how decoding fidelity impacts the rate-limiting steps of ribosome translocation.
• To explore the potential role of elongation factor P (EF-P) in mitigating translocation defects caused by miscoding.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) imaging was used to observe ribosome dynamics.
• Experiments involved introducing base-pairing mismatches between peptidyl-tRNA and mRNA codons.
• The effect of elongation factor P (EF-P) on translocation efficiency was assessed.

Main Results:

• Base-pairing mismatches between peptidyl-tRNA and mRNA significantly delay ribosome translocation.
• This delay is linked to the recognition and engagement of peptidyl-tRNA in the P site.
• Elongation factor P (EF-P) effectively rescues translocation defects caused by miscoding.

Conclusions:

• Ribosome translocation rate is sensitive to decoding accuracy, particularly peptidyl-tRNA recognition.
• Mis-coding antibiotics might impair protein synthesis by disrupting translocation.
• EF-P plays a broader role than previously known, assisting in resuming translation after miscoding errors.