Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by ~(15)N and ~2H ESEEM Spectroscopy

摘要

Although site-bound Mg~(2+) ions have been proposed to influence RNA structure and function,establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer.We have previously determined that,in solution,the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a K_(d,app) < 10 mu M for Mn~(2+) in 1 M NaCI and speculated that this site has functional importance in the ribozyme cleavage reaction.Here we determine both the precise location and the hydration level of Mn~(2+) in this site using ESEEM (electron spin-echo envelope modulation) spectroscopy.Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with ~(15)N guanine.The coordinated metal ion retains four water ligands as measured by ~2H ESEEM spectroscopy.The results presented here show that a functionally important,specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength.Although it has a relatively high thermodynamic affinity,this ion remains partially hydrated and is chelated to the RNA by just two ligands.