NMR structure of varkud satellite ribozyme stem-loop V in the presence of magnesium ions and localization of metal-binding sites.
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.
Campbell
Dean O
DO
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
Bouchard
Patricia
P
Desjardins
Geneviève
G
Legault
Pascale
P
D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000737:chemistry; D004722:Endoribonucleases / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology / Q000235:genetics; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D020871:RNA Stability; D016337:RNA, Catalytic / Q000737:chemistry*; D013329:Structure-Activity Relationship
D002413:Cations, Divalent; D016337:RNA, Catalytic; D004722:Endoribonucleases; C438195:varkud satellite ribozyme; D008274:Magnesium