Metal ion-N7 coordination in a ribozyme branch domain by NMR

Abstract

The N7 of purine nucleotides presents one of the most dominant metal ion binding sites in nucleic acids. However, the interactions between kinetically labile metal ions like Mg(2+) and these nitrogen atoms are inherently difficult to observe in large RNAs. Rather than using the insensitive direct (15)N detection, here we have used (2)J-[(1)H,(15)N]-HSQC (Heteronuclear Single Quantum Coherence) NMR experiments as a fast and efficient method to specifically observe and characterize such interactions within larger RNA constructs. Using the 27 nucleotides long branch domain of the yeast-mitochondrial group II intron ribozyme Sc.ai5gamma as an example, we show that direct N7 coordination of a Mg(2+) ion takes place in a tetraloop nucleotide. A second Mg(2+) ion, located in the major groove at the catalytic branch site, coordinates mainly in an outer-sphere fashion to the highly conserved flanking GU wobble pairs but not to N7 of the sandwiched branch adenosine.