Rüdisser S, Tinoco I
Department of Chemistry, University of California and Physical Biosciences Division Lawrence Berkeley National Laboratory, Berkeley, CA, 94720-1460, USA.
J Mol Biol. 2000 Feb 4;295(5):1211-23. doi: 10.1006/jmbi.1999.3421.
The solution structure of a 22 nt RNA hairpin and its complex with Co(NH(3))(6)(3+) bound to the GAAA tetraloop has been determined by NMR spectroscopy. Co(NH(3))(6)(3+) has a similar geometry to Mg(H(2)O)(6)(2+) and can be used as a probe for binding sites of completely solvated magnesium ions. The hairpin contains tandem G.A mismatches, similar to the P5abc region of a group I intron, and is closed by a GAAA tetraloop. The tandem G.A mismatches are imino hydrogen bonded in contrast with the sheared G.A mismatches found in a different context in the crystal structure of the P4-P6 domain. Chemical shift changes of the imino protons upon titration of the RNA hairpin with Mg(2+) and with Co(NH(3))(6)(3+) were used to identify ion-binding sites. Paramagnetic resonance broadening upon titration with Mn(2+) was also used. The titration curves gave dissociation binding constants for the magnesium ions in the millimolar range, similar to the binding in the major groove of RNA at tandem G.U base-pairs. Although the largest chemical shift change occurred at an imino proton of one of the G.A base-pairs, no nuclear Overhauser enhancement cross-peaks between the cobalt ligand and neighboring RNA protons were seen, presumably due to the high mobility of the Co(NH(3))(6)(3+) at this site. Nuclear Overhauser enhancement cross-peaks between Co(NH(3))(6)(3+) and the GAAA tetraloop were observed, which allowed the determination of the structure of the tetraloop binding site. The Co(NH(3))(6)(3+) is bound in the major groove of the GAAA tetraloop with hydrogen bonds to guanine base N7 and to phosphate oxygen atoms of the tetraloop.
通过核磁共振光谱法测定了一个22个核苷酸的RNA发夹结构及其与结合在GAAA四环上的六氨合钴（III）（Co(NH(3))(6)(3+)）形成的复合物的结构。六氨合钴（III）（Co(NH(3))(6)(3+)）具有与六水合镁（II）（Mg(H(2)O)(6)(2+)）相似的几何结构，可作为完全溶剂化镁离子结合位点的探针。该发夹包含串联的G.A错配，类似于I组内含子的P5abc区域，并由一个GAAA四环封闭。与在P4-P6结构域晶体结构中不同环境下发现的剪切G.A错配相反，串联的G.A错配通过亚氨基氢键连接。在用镁离子（Mg(2+)）和六氨合钴（III）（Co(NH(3))(6)(3+)）滴定RNA发夹时，亚氨基质子的化学位移变化用于识别离子结合位点。用锰离子（Mn(2+)）滴定引起的顺磁共振加宽也被使用。滴定曲线给出了毫摩尔范围内镁离子的解离结合常数，类似于RNA在串联G.U碱基对处大沟中的结合情况。尽管最大的化学位移变化发生在其中一个G.A碱基对的亚氨基质子上，但在钴配体与相邻RNA质子之间未观察到核Overhauser增强交叉峰，推测是由于该位点的六氨合钴（III）（Co(NH(3))(6)(3+)）具有高流动性。观察到六氨合钴（III）（Co(NH(3))(6)(3+)）与GAAA四环之间的核Overhauser增强交叉峰，这使得能够确定四环结合位点的结构。六氨合钴（III）（Co(NH(3))(6)(3+)）通过与鸟嘌呤碱基N7和四环的磷酸氧原子形成氢键，结合在GAAA四环的大沟中。
