Nucleic Acids Research, 2002, Vol. 30, No. 23 5151-5159
© 2002 Oxford University Press
Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme
1 Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260-3000, USA and 2 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
*To whom correspondence should be addressed. Tel: +1 716 645 6800; Fax: +1 716 645 6963; Email: hsuga{at}acsu.buffalo.edu
Present address:
Hirohide Saito, Department of Protein Engineering, Cancer Institute, Japanese Foundation for Cancer Research, Toshima, Tokyo 170-8455, Japan
Metal ions are essential cofactors for various ribozymes. Here we dissect the roles of metal ions in an aminoacyl-tRNA synthetase-like ribozyme (ARS ribozyme), which was evolved in vitro. This ribozyme can charge phenylalanine on tRNA in cis, where it is covalently attached to the 5'-end of tRNA (i.e. a form of precursor tRNA), as well as in trans, where it can act as a catalyst. The presence of magnesium ion is essential for this ribozyme to exhibit full catalytic activity. Metal-dependent kinetics, as well as structural mappings using Tb3+ in competition with Mg2+ or Co(NH3)63+, identified two potential metal-binding sites which are embedded near the tRNA-binding site. The high affinity metal-binding site can be filled with either Mg2+ or Co(NH3)63+ and thus the activity relies on a metal ion that is fully coordinated with water or ammonium ions. This site also overlaps with the amino acid-binding site, suggesting that the metal ion plays a role in constituting the catalytic core. The weak metal-binding site is occupied only by a metal ion(s) that can form innersphere contacts with ligands in the ribozyme and, hence, Mg2+ can enhance ribozyme activity, but Co(NH3)63+ cannot. The experiments described in this work establish the roles of metal ions that have distinct coordination properties in the ARS ribozyme.