NMR Structure of Varkud Satellite Ribozyme Stem-Loop V in the Presence of Magnesium Ions and Localization of Metal-Binding Sites

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• 作者：Dean O. Campbell ; Patricia Bouchard ; Genevi&egrave ; ve Desjardins ; Pascale Legault
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：September 5, 2006
• 年：2006
• 卷：45
• 期：35
• 页码：10591 - 10605
• 全文大小：778K
• 年卷期：v.45,no.35(September 5, 2006)
• ISSN：1520-4995

文摘

In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interactionbetween 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 Mg²⁺(SL5^Mg) and demonstrate that Mg²⁺ induces a conformational change in which the SL5 loop adopts acompact structure with most characteristics of canonical U-turn structures. Divalent cation-binding siteswere probed with Mn²⁺-induced paramagnetic line broadening and intermolecular NOEs to Co(NH₃)₆³⁺.Structural modeling of Mn(H₂O)₆²⁺ in SL5^Mg revealed four divalent cation-binding sites in the loop. Sites1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent toN7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 arepresent in other U-turn motifs, and these metal-binding sites may represent a common feature of theU-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change theconformation of residues 697-699 involved in the proposed Watson-Crick base pairs with stem-loopI. In both the presence and the absence of Mg²⁺, G697, A698, and C699 adopt an A-form structure thatexposes 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 proximityof the bases of G697, A698, and C699, suggesting that the Mg²⁺-bound conformation of stem-loop Vallows additional contacts with stem-loop I. These studies improve our understanding of the role ofMg²⁺ in U-turn structures and in substrate recognition by the VS ribozyme.