Dynamic RNA–protein interactions underpin numerous molecular control mechanisms in biology. However, relatively little is known about the kinetic landscape of protein interactions with full-length RNAs. The extent to which interaction kinetics vary for the same RNA element across the transcriptome and the molecular determinants of variability therefore remain poorly defined. Moreover, it is unclear how one protein–RNA interaction might be transduced by RNA to kinetically impact a second. We report a parallelized, real-time single-molecule fluorescence assay for protein interaction kinetics on eukaryotic mRNA populations obtained from cells. We observed ∼100-fold heterogeneity for interactions of the translation initiation factor eIF4E with the universal mRNA 5′ cap structure, dominated by steric effects on barrier-height variability for association. We also found that an RNA helicase, eIF4A, independently accelerated eIF4E–cap association. These data support a kinetic mechanism for how mRNA can determine the sensitivity of its translation to reduction in cellular eIF4E concentrations. They also support the view that global RNA structure significantly modulates protein–RNA interaction dynamics and can facilitate real-time communication between protein interactions at distinct sites.
nucleation, the process by which microtubules nucleate off the sides of preexisting microtubules. This autocatalytic nucleation pathway is critical for spindle assembly in eukaryotic cells. Using fluorescence, electron, and atomic force microscopies and hydrodynamic theory, we show that TPX2 on a microtubule reorganizes according to the Rayleigh-Plateau instability. This is the same phenomenon that causes a film of dew to form droplets along a spider web. After uniformly coating microtubules, TPX2 forms regularly spaced droplets from which branches nucleate. Droplet size and spacing increase with greater TPX2 concentration. A stochastic model shows that droplets make branching nucleation more efficient by confining the space along the microtubule where multiple necessary factors colocalize to nucleate a branch. Our work is the first demonstration of the Rayleigh-Plateau instability in nanoscale molecular biology. Although we study it in the context of branching nucleation, it is a generic process that only requires a condensed phase to coat a filament. Thus, we anticipate that similar hydrodynamic effects may play important roles in other areas wherever condensed proteins and biological filaments meet.
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