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

摘要

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 interaction makes to the folding properties of a RNA.To investigate the kinetics and thermodynamics for the isolated interaction,a GAAA tetraloop domain and receptor domain were connected by a single-stranded A_7 linker.Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular docking of the GAAA tetraloop and receptor.Docking was induced using a variety of metal ions,where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking {[Co(NH_3)_6~(3+)] " [Ca~(2+)],[Mg~(2+)],[Mn~(2+)] " [Na~+],[K~+]}.Analysis of metal ion cooperativity yielded Hill coefficients of approx 2 for Na~+- or K~+-dependent docking versus approx 1 for the divalent ions and Co(NH_3)_6~(3+).Ensemble stopped-flow FRET kinetic measurements yielded an apparent activation energy of 12.7 kcal/mol for GAAA tetraloop-receptor docking.RNA constructs with U_7 and A_(14) single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine how linker length and composition affect docking.These studies showed that the single-stranded region functions primarily as a flexible tether.Inhibition of docking by oligonucleotides complementary to the linker was also investigated.The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking is discussed.