Abstract

Previous studies showed that cytoplasmic and mitochondrial forms of yeast valyl-tRNA synthetase (ValRS) are specified by the VAS1 gene through alternative initiation of translation. Sequence comparison suggests that the yeast cytoplasmic (or mature mitochondrial) ValRS contains an N-terminal appendage that acts in cis as a nonspecific tRNA-binding domain (TRBD) and is absent from its bacterial relatives. We show here that Escherichia coli ValRS can substitute for the mitochondrial and cytoplasmic functions of VAS1 by fusion of a mitochondrial targeting signal and a TRBD, respectively. In addition, the bacterial ValRS gene can be converted into a dual functional yeast gene encoding both cytoplasmic and mitochondrial activities by fusion of a DNA sequence specifying both the mitochondrial targeting signal and TRBD. In vitro assays suggested that fusion of a nonspecific TRBD to the bacterial enzyme significantly enhanced its yeast tRNA-binding and aminoacylation activities. These results not only underscore the necessity of retaining a TRBD for functioning of a tRNA synthetase in yeast cytoplasm, but also provide insights into the evolution of tRNA synthetase genes.