Docking kinetics and equilibrium of a GAAA tetraloop-receptor motif probed by single-molecule FRET

JOSE H. HODAK; CHRISTOPHER D. DOWNEY; JULIE L. FIORE; ARTHUR PARDI; DAVID J. NESBITT

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA

Año: 2005 vol. 102 p. 10505 - 10510

0027-8424

Docking kinetics and equilibrium of fluorescently labeled RNAmolecules are studied with single-molecule FRET methods. TimeresolvedFRET is used to monitor docking-undocking transitions forRNAs containing a single GAAA tetraloop-receptor tertiary interactionconnected by a flexible single-stranded linker. The rateconstants for docking and undocking are measured as a function ofMg2+, revealing a complex dependence on metal ion concentration.Despite the simplicity of this model system, conformationalheterogeneity similar to that noted in more complex RNA systemsis observed; relatively rapid docking-undocking transitions aredetected for approximately two-thirds of the RNA molecules, withsignificant subpopulations exhibiting few or no transitions on the10- to 30-s time scale for photobleaching. The rate constants aredetermined from analysis of probability densities, which allows amuch wider range of time scales to be analyzed than standardhistogram procedures. The data for the GAAA tetraloop receptorare compared with kinetic and equilibrium data for other RNAtertiary interactions.RNA  tertiary interactions