Co-temporal Force and Fluorescence Measurements Reveal a Ribosomal Gear Shift Mechanism of Translation Regulation by...

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• Ribosome movement on mRNA is crucial for protein synthesis.
• mRNA secondary structures act as mechanical barriers, regulating translation.
• The role of elongation factor G (EF-G) in overcoming these barriers is not fully understood.

Purpose of the Study:

• To investigate how ribosomes, with the help of EF-G, unwind mRNA secondary structures.
• To elucidate the coupling between ribosome conformational changes and translocation factor activity.
• To understand the ribosome's response to mechanical barriers during translation.

Main Methods:

• High-resolution optical tweezers were used to apply force to mRNA.
• Single-molecule fluorescence was employed to monitor ribosome dynamics.
• The mechanical properties of mRNA hairpin structures were modulated.

Main Results:

• Hairpin opening during EF-G-catalyzed translocation is driven by small subunit head rotation.
• Ribosomes shift to a 7-fold slower kinetic pathway when encountering strong barriers.
• This shift involves an allosteric switch, potentially utilizing thermal fluctuations.
• A novel two-step sub-codon hairpin opening mechanism was observed.

Conclusions:

• Ribosomes actively unwind mRNA secondary structures via EF-G-mediated translocation.
• The ribosome exhibits adaptive mechanisms, including a slower kinetic pathway, to overcome mechanical stress.
• These findings reveal new insights into translation regulation and ribosome mechanics.