Campbell Dean O, Bouchard Patricia, Desjardins Geneviève, Legault Pascale
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Biochemistry. 2006 Sep 5;45(35):10591-605. doi: 10.1021/bi0607150.
In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interaction between stem-loops I and V, which is important for recognition and activation of the stem-loop I substrate. Here, we present the high-resolution NMR structure of stem-loop V (SL5) in the presence of Mg(2+) (SL5(Mg)) and demonstrate that Mg(2+) induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn(2+)-induced paramagnetic line broadening and intermolecular NOEs to Co(NH(3))(6)(3+). Structural modeling of Mn(H(2)O)(6)(2+) in SL5(Mg) revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697-699 involved in the proposed Watson-Crick base pairs with stem-loop I. In both the presence and the absence of Mg(2+), G697, A698, and C699 adopt an A-form structure that exposes their Watson-Crick faces, and this is compatible with their proposed interaction with stem-loop I. In SL5(Mg), however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg(2+)-bound conformation of stem-loop V allows additional contacts with stem-loop I. These studies improve our understanding of the role of Mg(2+) in U-turn structures and in substrate recognition by the VS ribozyme.
在粗糙脉孢菌VS核酶中，镁离子促进茎环I和V之间形成环-环相互作用，这对于识别和激活茎环I底物很重要。在此，我们展示了在Mg(2+)存在下茎环V（SL5）的高分辨率核磁共振结构（SL5(Mg)），并证明Mg(2+)诱导了一种构象变化，其中SL5环采用了具有典型U型转弯结构大多数特征的紧凑结构。用Mn(2+)诱导的顺磁线宽化和与Co(NH3)6(3+)的分子间核Overhauser效应探测二价阳离子结合位点。SL5(Mg)中Mn(H2O)6(2+)的结构建模揭示了环中有四个二价阳离子结合位点。位点1、3和4位于靠近多个磷酸基团的大沟中，而位点2在小沟中与G697的N7和A698的N7相邻。其他U型转弯基序中存在与SL5中位点1 - 3等效的阳离子结合位点，这些金属结合位点可能代表了U型转弯折叠的一个共同特征。尽管镁离子影响环的构象，但它们不会显著改变与茎环I形成拟沃森-克里克碱基对的697 - 699位残基的构象。在有和没有Mg(2+)的情况下，G697、A698和C699都采用A构象结构，暴露它们的沃森-克里克面，这与它们与茎环I的拟相互作用是一致的。然而，在SL5(Mg)中，U700在环的小沟面上暴露于G697、A698和C699碱基附近，这表明茎环V的Mg(2+)结合构象允许与茎环I进行额外的接触。这些研究增进了我们对Mg(2+)在U型转弯结构以及VS核酶识别底物中的作用的理解。
