title: C-terminal zinc-containing peptide required for RNA recognition by a class I tRNA synthetase.
abstract: Escherichia coli isoleucyl-tRNA synthetase is one of five closely related class I tRNA synthetases. The active site of the 939 amino acid polypeptide is in an N-terminal domain which contains an insertion believed essential for interactions with the tRNA acceptor helix. The enzyme was shown previously to contain an essential (for function in vivo) zinc bound to a Cys4 cluster at the C-terminal end of the polypeptide. The specific function of this zinc has been unknown. We show here that aminoacylation activity can be reconstituted in vitro by combining a 53 amino acid zinc-containing C-terminal peptide with a protein consisting of the remaining 886 amino acids. Reconstitution of aminoacylation is zinc-dependent. In contrast, the zinc-containing peptide is dispensable for synthesis of isoleucyl adenylate. Affinity coelectrophoresis showed that the 53 amino acid C-terminal peptide is required specifically for tRNA binding. We propose that the zinc-containing peptide curls back to the active site to make contact with the acceptor helix of bound tRNA, but not with isoleucine or ATP. It is the first example of a zinc-containing peptide in a class I tRNA synthetase that is essential for tRNA binding interactions. The design of this enzyme may be part of a more general scheme for class I tRNA synthetases to acquire acceptor helix binding elements during the development of the genetic code.
authors.affiliation: Department of Biology, Massachusetts Institute of Technology, Cambridge, 02139, USA.
authors.forename: E
authors.initials: E
authors.lastname: Glasfeld
authors.forename: J A
authors.initials: JA
authors.lastname: Landro
authors.forename: P
authors.initials: P
authors.lastname: Schimmel
mesh_terms: D000595:Amino Acid Sequence; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase; D015032:Zinc