C-TERMINAL ZINC-CONTAINING PEPTIDE REQUIRED FOR RNA RECOGNITION BY A CLASS-I TRANSFER-RNA SYNTHETASE

Escherichia coil isoleucyl-tRNA synthetase is one of five closely rela ted class I tRNA synthetases. The active site of the 939 amino acid po lypeptide is in an N-terminal domain which contains an insertion belie ved essential for interactions with the tRNA acceptor helix. The enzym e was shown previously to contain an essential (for function in vivo) zinc bound to a Cys(4) cluster at the C-terminal end of the polypeptid e. The specific function of this zinc has been unknown. We show here t hat aminoacylation activity can be reconstituted in vitro by combining a 53 amino acid zinc-containing C-terminal peptide with a protein con sisting of the remaining 886 amino acids. Reconstitution of aminoacyla tion is zinc-dependent. In contrast, the zinc-containing peptide is di spensable for synthesis of isoleucyl adenylate. Affinity coelectrophor esis showed that the 53 amino acid C-terminal peptide is required spec ifically 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 exampl e of a zinc-containing peptide in a class I tRNA synthetase that is es sential for tRNA binding interactions. The design of this enzyme may b e part of a more general scheme for class I tRNA synthetases to acquir e acceptor helix binding elements during the development of the geneti c code.