Nucleic Acids Research, 2001, Vol. 29, No. 11 2401-2408
© 2001 Oxford University Press
Association of an RNA kissing complex analyzed using 2-aminopurine fluorescence
Center for Advanced Research in Biotechnology of the University of Maryland and the National Institute for Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850, USA
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 kissing 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 (Fmax = 370 nm), which provides a sensitive measure for the binding reaction. The 2-AP substituted complexes are found to have equilibrium binding properties (average KD = 2.6 ± 1.7 nM) and affinity for Rom (average KD = 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 koff = 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 µM–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.
* To whom correspondence should be addressed. Tel: +1 301 738 6160; Fax: +1 301 738 6255; Email: marino{at}carb.nist.gov
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