Metal Ion Dependence, Thermodynamics, and Kinetics for Intramolecular Docking of a GAAA Tetraloop and Receptor Connected by a Flexible Linker

• Published in: Biochemistry (Easton) Vol. 45; no. 11; pp. 3664 - 3673
• Main Authors: Downey, Christopher D, Fiore, Julie L, Stoddard, Colby D, Hodak, Jose H, Nesbitt, David J, Pardi, Arthur
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 21.03.2006
• Subjects: Base Sequence; Cations - chemistry; Dose-Response Relationship, Drug; Fluorescence Resonance Energy Transfer; Kinetics; Models, Biological; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotides - chemistry; Oligonucleotides - metabolism; RNA - chemistry; RNA - metabolism; Thermodynamics; Time Factors
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0520941

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 A7 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(NH3)6 3+] ≪ [Ca2+], [Mg2+], [Mn2+] ≪ [Na+], [K+]}. Analysis of metal ion cooperativity yielded Hill coefficients of ≈2 for Na+- or K+-dependent docking versus ≈1 for the divalent ions and Co(NH3)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 U7 and A14 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.