Metal Ion Dependence, Thermodynamics, and Kinetics for Intramolecular Docking of a GAAA Tetraloop and Receptor Connected by a Flexible Linker
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- 作者：Christopher D. Downey ; Julie L. Fiore ; Colby D Stoddard ; Jose H. Hodak ; David J. Nesbitt ; and Arthur Pardi
- 刊名：Biochemistry
- 出版年：2006
- 出版时间：March 21, 2006
- 年：2006
- 卷：45
- 期：11
- 页码：3664 - 3673
- 全文大小：370K
- 年卷期：v.45,no.11(March 21, 2006)
- ISSN：1520-4995
文摘
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 interactionmakes to the folding properties of a RNA. To investigate the kinetics and thermodynamics for the isolatedinteraction, a GAAA tetraloop domain and receptor domain were connected by a single-stranded A7 linker.Fluorescence resonance energy transfer (FRET) experiments were used to probe intramolecular dockingof the GAAA tetraloop and receptor. Docking was induced using a variety of metal ions, where thecharge of the ion was the most important factor in determining the concentration of the ion required topromote docking {[Co(NH3)63+] 
[Ca2+], [Mg2+], [Mn2+] [Na+], [K+]}. Analysis of metal ioncooperativity yielded Hill coefficients of 
2 for Na+- or K+-dependent docking versus 1 for the divalentions and Co(NH3)63+. Ensemble stopped-flow FRET kinetic measurements yielded an apparent activationenergy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U7 and A14 single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine howlinker length and composition affect docking. These studies showed that the single-stranded region functionsprimarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker wasalso investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking isdiscussed.
[Ca2+], [Mg2+], [Mn2+] [Na+], [K+]}. Analysis of metal ioncooperativity yielded Hill coefficients of 2 for Na+- or K+-dependent docking versus 1 for the divalentions and Co(NH3)63+. Ensemble stopped-flow FRET kinetic measurements yielded an apparent activationenergy of 12.7 kcal/mol for GAAA tetraloop-receptor docking. RNA constructs with U7 and A14 single-stranded linkers were investigated by single-molecule and ensemble FRET techniques to determine howlinker length and composition affect docking. These studies showed that the single-stranded region functionsprimarily as a flexible tether. Inhibition of docking by oligonucleotides complementary to the linker wasalso investigated. The influence of flexible versus rigid linkers on GAAA tetraloop-receptor docking isdiscussed.