Cellular Sticking Can Strongly Reduce Complex Binding by Speeding Dissociation.

Area of Science:

• Molecular Biology
• Biophysics
• Cell Biology

Background:

• In vitro studies often overestimate protein-RNA binding affinity.
• Cellular environments contain numerous nonspecific interactions.
• The effect of the cellular environment on protein-RNA binding dynamics is not well understood.

Purpose of the Study:

• To experimentally determine the role of the cellular environment on protein-RNA binding affinity and dynamics.
• To compare in vitro and in vivo binding parameters for a specific protein-RNA interaction.

Main Methods:

• Utilized a Förster resonance energy transfer (FRET) based assay in living U-2 OS cells.
• Employed a laser-induced temperature jump to perturb binding equilibrium.
• Quantified association and dissociation rate coefficients in vitro and in vivo.

Main Results:

• Apparent protein-RNA binding affinity in cells was reduced by up to two orders of magnitude compared to in vitro.
• In-cell dissociation rate coefficients were up to two orders of magnitude larger.
• Association rate coefficients showed no significant change in the cellular environment.

Conclusions:

• Nonspecific interactions in the cellular environment significantly reduce effective protein-RNA binding affinity.
• High in vitro binding affinities are necessary for robust protein-RNA interactions in vivo.
• Cellular components can bind protein-RNA complexes, but with reduced affinity.