Molecular crowding stabilizes folded RNA structure by the excluded volume effect.

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Macromolecular folding is influenced by solution conditions, including the presence of crowder molecules.
• Understanding RNA folding in vivo requires accounting for molecular crowding effects.
• Polyethylene glycol (PEG) is a common molecular crowder used to study these effects.

Purpose of the Study:

• To investigate the impact of polyethylene glycol 1000 (PEG-1000) on the folding of a bacterial group I ribozyme.
• To quantify the excluded volume effect of PEG-1000 on RNA structure.
• To determine how PEG-1000 influences the thermodynamics of RNA folding.

Main Methods:

• Small angle X-ray scattering (SAXS) experiments were performed on a 64 kDa bacterial group I ribozyme.
• Experiments were conducted in the presence of varying concentrations of PEG-1000 (0-20% wt/vol).
• Changes in water and ion activities were measured, and theoretical models were applied to assess excluded volume effects.

Main Results:

• PEG-1000 was found to favor more compact RNA structures, consistent with excluded volume effects.
• The transition to the folded state occurred at lower MgCl(2) concentrations in the presence of PEG.
• The radius of gyration of the unfolded RNA decreased from 76 to 64 Å as PEG concentration increased.

Conclusions:

• The dominant effect of PEG-1000 as a molecular crowder on RNA folding is the excluded volume effect.
• PEG-1000 promotes compact RNA conformations by sterically hindering unfolded states.
• These findings highlight the importance of considering molecular crowding in cellular RNA folding processes.