Association of an RNA Kissing Complex Analyzed Using 2-Aminopurine Fluorescence

Published

June 1, 2001

Author(s)

M J. Rist, John Marino

Abstract

The fluorescent probe, 2-aminopurine-2'-O-methyl riboside (2-AP) has been selectively incorporated at adenosine positions in stem-loops (so called R1inv and R2inv), derived from the ColE1 plasmid encoded RNA I and RNA II transcripts, that interact to form stable loop-loop kissing complexes and bind the RNA one modulator (Rom) protein, such that fluorescence-detected stopped-flow and equilibrium methods could be used to study the detailed mechanism of this RNA-RNA interaction. Formation of loop-loop complexes between R1inv and R2inv hairpins, substituted with 2-AP at positions in the complementary loops, results in a 5 -10-fold fluorescence emission decrease (F_max = 370 nm), which provides a sensitive measure for the binding reaction. The 2-AP substituted complexes are found to have equilibrium binding properties (average K_D = 2.6 1.7 nM) and affinity for ROM (average K_D = 60 24 nM) that are similar to complexes formed with equivalent unlabeled hairpins. Using stopped-flow experiments, it was found that the 2-AP probes experienced at least three different microenvironments during association of the RNA complex, thus suggesting a kinetic intermediate in the kissing pathway. In contrast, dissociation of the complex was found to fit a single exponential decay (average K_off = 8.9 X 10^-5 S^-1). Consistent with these observations, a two-step mechanism for RNA loop -loop complex association is proposed in which the complementary loops of R1inv and R2inv first base pair to form the loop-loop helix (average k₁ = 0.13 M^-1s^-1) in the initial encounter reaction, and subsequently isomerize to the final tertiary fold in a second slower step (average k₂ = 0.09 s^-1), where the helical stacking around the junctions is optimized.

Citation

Nucleic Acids Research

Volume

29

Issue

No. 11

Pub Type

Journals

Keywords

2-aminopurine, antisense, ColE1, fluorescence, RNA

Citation

Rist, M. and Marino, J. (2001), Association of an RNA Kissing Complex Analyzed Using 2-Aminopurine Fluorescence, Nucleic Acids Research (Accessed December 11, 2024)