Transient disome complex formation in native polysomes during ongoing protein synthesis captured by cryo-EM

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Bacterial translation research traditionally uses in vitro assembly and ribosome stalling.
• Visualizing actively elongating polysomes provides a more dynamic view of translation.

Purpose of the Study:

• To visualize actively elongating bacterial polysomes using cryo-electron microscopy (cryo-EM).
• To characterize the functional states and structural dynamics of disome complexes during translation elongation.

Main Methods:

• Reactivation of ex vivo-derived E. coli polysomes in the PURE in vitro translation system.
• Cryo-electron microscopy (cryo-EM) analysis of actively elongating polysomes.
• RNase digest experiments to investigate complex formation.

Main Results:

• 31% of 70S ribosomes formed disome complexes, representing eight distinct functional states.
• Paused disome complexes were observed to form transiently during ongoing elongation.
• Five disome interfaces were identified, showing rearrangements during the elongation cycle.
• bL9's CTD was found to obstruct the factor binding site of queueing ribosomes, preventing collisions.

Conclusions:

• Actively elongating polysomes exhibit diverse functional states and transient pausing.
• Ribosome-ribosome contacts dynamically rearrange during translation elongation.
• Structural mechanisms, like bL9 CTD obstruction, prevent harmful ribosome collisions.