Metal ion dependence, thermodynamics, and kinetics for intramolecular docking of a GAAA tetraloop and receptor connected by a flexible linker.
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 approximately 2 for Na(+)- or K(+)-dependent docking versus approximately 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.
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Christopher D
CD
Downey
Julie L
JL
Fiore
Colby D
CD
Stoddard
Jose H
JH
Hodak
David J
DJ
Nesbitt
Arthur
A
Pardi
D001483:Base Sequence; D002412:Cations / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D013997:Time Factors
D002412:Cations; D009841:Oligonucleotides; D012313:RNA