Metal Ion Dependence, Thermodynamics, and Kinetics for Intramolecular Docking of a GAAA Tetraloop and Receptor Connected by a Flexible Linker

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

BIOCHEMISTRY

AMER CHEMICAL SOC

Año: 2006 vol. 45 p. 3664 - 3673

0006-2960

The GAAA tetraloop-receptor motif is a commonly occurring tertiary interaction in RNA.This motif usually occurs in combination with other tertiary interactions in complex RNA structures.Thus, it is difficult to measure directly the contribution that a single GAAA tetraloop-receptor interactionmakes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolatedinteraction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A7 linker.Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular dockingof the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where thecharge of the ion was the most important factor in determining the concentration of the ion required topromote docking {[Co(NH3)63+] , [Ca2+], [Mg2+], [Mn2+] , [Na+], [K+]}. Analysis of metal ioncooperativity yielded Hill coefficients of 2 for Na+- or K+-dependent docking versus 1 for the divalentions and Co(NH3)63+. Ensemble stopped-flow FRET kinetic measurements yielded an apparent activationenergy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U7 and A14 singlestrandedlinkers were investigated by single-molecule and ensemble FRET techniques to determine howlinker length and composition affect docking. These studies showed that the single-stranded region functionsprimarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker wasalso investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking isdiscussed.