Downey Christopher D, Fiore Julie L, Stoddard Colby D, Hodak Jose H, Nesbitt David J, Pardi Arthur
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA.
Biochemistry. 2006 Mar 21;45(11):3664-73. 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 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.
GAAA四环-受体基序是RNA中常见的三级相互作用。该基序通常与复杂RNA结构中的其他三级相互作用共同出现。因此，很难直接测量单个GAAA四环-受体相互作用对RNA折叠特性的贡献。为了研究这种孤立相互作用的动力学和热力学，通过单链A(7)接头连接了一个GAAA四环结构域和受体结构域。荧光共振能量转移(FRET)实验用于探测GAAA四环与受体的分子内对接。使用多种金属离子诱导对接，其中离子电荷是决定促进对接所需离子浓度的最重要因素{[Co(NH(3))(6)(3+)] << [Ca(2+)], [Mg(2+)], [Mn(2+)] << [Na(+)], [K(+)]}。对金属离子协同性的分析得出，对于依赖Na(+)或K(+)的对接，希尔系数约为2，而对于二价离子和Co(NH(3))(6)(3+)，希尔系数约为1。整体停流FRET动力学测量得出GAAA四环-受体对接的表观活化能为12.7 kcal/mol。通过单分子和整体FRET技术研究了具有U(7)和A(14)单链接头的RNA构建体，以确定接头长度和组成如何影响对接。这些研究表明，单链区域主要起柔性系链的作用。还研究了与接头互补的寡核苷酸对对接的抑制作用。讨论了柔性接头与刚性接头对GAAA四环-受体对接的影响。
