ZINC-DEPENDENT CELL-GROWTH CONFERRED BY MUTANT TRANSFER-RNA SYNTHETASE

We present evidence that zinc bound near the C terminus of a long tRNA synthetase polypeptide, and at a location far in the sequence from th e catalytic domain, is needed to sustain cell growth and is, therefore , essential for enzyme function. Several class I and class II tRNA syn thetases contain bound zinc, including the 939-amino acid class I Esch erichia coli isoleucyl-tRNA synthetase, which has two zinc atoms coord inated to cysteine sulfhydryls. The functional significance of these b ound zinc atoms has been unclear. Like other class I tRNA synthetases, the isoleucine enzyme has a class-defining conserved N-terminal domai n that contains the catalytic site. The C-terminal domain is variable in sequence and structure and not conserved among all of the class I e nzymes. Using split proteins, we localized a zinc binding site to the C-terminal end of isoleucyl-tRNA synthetase. Serine substitutions of s ingle cysteines at a thiol-containing putative zinc binding site that is less than 40 amino acids from the C terminus confer a zinc-dependen t growth phenotype on cells harboring the mutant enzymes. We propose t hat zinc bound near the C terminus is part of a structure that interac ts directly or indirectly with the active site. A structure at the C t erminus that provides a functional link between the conserved N-termin al catalytic and non-conserved C-terminal domain may be common to seve ral class I enzymes.