Association of an RNA kissing complex analyzed using 2-aminopurine fluorescence

The fluorescent probe, 2-aminopurine-2'-O-methyl riboside (2-AP) has been s electively incorporated at adenosine positions in stem-loops (so called R1i nv and R2inv), derived from the ColE1 plasmid encoded RNA I and RNA II tran scripts, that interact to form stable loop-loop kissing complexes and bind the RNA one modulator (Rom) protein, such that fluorescence-detected stoppe d-flow and equilibrium methods could be used to study the detailed mechanis m of this RNA-RNA interaction. Formation of loop-loop kissing complexes bet ween R1inv and R2inv hairpins, substituted with 2-AP at positions in the co mplementary 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 prop erties (average K-D = 2.6 +/- 1.7 nM) and affinity for Rom (average K-D = 6 0 +/- 24 nM) that are similar to complexes formed with equivalent unlabeled hairpins, Using stopped-flow experiments, it was found that the 2-AP probe s 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 t hese observations, a two-step mechanism for RNA loop-loop complex associati on is proposed in which the complementary loops of R1inv and R2inv first ba se pair to form the loop-loop helix (average k(1) = 0.13 muM(-1)s(-1)) in t he initial encounter reaction, and subsequently isomerize to the final tert iary fold in a second slower step (average k(2)= 0.09 s(-1)), where the hel ical stacking around the junctions is optimized.