On the pH dependence of class-1 RF-dependent termination of mRNA translation

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Bacterial protein synthesis termination is crucial for cellular function.
• Class-1 release factors (RF1 and RF2) mediate stop codon recognition and polypeptide release.
• The pH dependence of this process is not fully understood.

Purpose of the Study:

• To investigate the pH dependence of the termination rate catalyzed by class-1 release factors (RF1/RF2).
• To elucidate the rate-limiting steps in RF-mediated termination at different pH values.
• To understand the role of hydroxide ions versus water molecules in ester bond hydrolysis.

Main Methods:

• Utilized classical quench-flow techniques.
• Employed a novel stopped-flow method with fluorescently labeled peptides.
• Analyzed the pH dependence of termination rates and determined apparent pKa values.

Main Results:

• Termination rate increased with pH, saturating at approximately 70 s⁻¹.
• An apparent pKa of ~7.6 was observed.
• Identified distinct rate-limiting steps: ester bond hydrolysis at low pH and a conformational change at high pH.

Conclusions:

• Class-1 RF termination is governed by ester bond hydrolysis at low pH and a stop-codon-dependent conformational change at high pH.
• Propose that hydroxide ions, rather than water, participate in the hydrolysis step.
• Offer an explanation for observed saturation differences in termination rates across studies.