Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme.
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)6(3+), 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)6(3+) 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)6(3+) cannot. The experiments described in this work establish the roles of metal ions that have distinct coordination properties in the ARS ribozyme.
Saito
Hirohide
H
Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260-3000, USA.
Suga
Hiroaki
H
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002412:Cations; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D008670:Metals / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D009690:Nucleic Acid Conformation; D010649:Phenylalanine / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D013725:Terbium / Q000378:metabolism / Q000494:pharmacology
D002412:Cations; D008670:Metals; D012322:RNA Precursors; D016337:RNA, Catalytic; D013725:Terbium; D010649:Phenylalanine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D008274:Magnesium