Cotemporal Single-Molecule Force and Fluorescence Measurements to Determine the Mechanism of Ribosome Translocation

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• Ribosomes are essential molecular machines translating genetic code into proteins.
• Protein synthesis involves complex mechanochemical processes, including mRNA translocation.
• Understanding ribosome mechanics is key to gene expression.

Purpose of the Study:

• To describe methods for real-time monitoring of ribosome function.
• To investigate how ribosomes use chemical energy for mechanical work.
• To uncover the mechanism of force generation during translation.

Main Methods:

• Utilized a high-resolution optical tweezers instrument with single-molecule fluorescence (fleezers).
• Monitored two orthogonal channels: elongation factor G (EF-G) binding and translocation.
• Studied single, actively translating ribosomes in real-time.

Main Results:

• Detailed procedures for monitoring ribosome EF-G binding and translocation are presented.
• The study provides insights into the mechanochemical coupling of ribosome movement.
• Real-time data reveals how chemical energy drives mechanical displacement.

Conclusions:

• The described methods enable detailed mechanistic studies of ribosome dynamics.
• This work elucidates how ribosomes generate mechanical force during protein synthesis.
• Understanding these processes is crucial for comprehending gene expression.