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.
Highlights
In some cases, cytoplasmic and mitochondrial forms of a tRNA synthetase with a given amino acid specificity are encoded by the same nuclear gene through alternative initiation of translation, examples of which include ALA1 [6, 7], GRS1 [8], HTS1 [9], and VAS1 (coding for valyl-tRNA synthetase (ValRS)) [10]
In addition to serving as a cis-acting tRNAbinding domain (TRBD), the appended domain (Ad) of some yeast tRNA synthetases were found to participate in protein-protein interactions, such as those of yeast glutamyl, methionyl- [20], and seryltRNA synthetases [21]
We focused on the cross-species and crosscompartmental complementation activities of a bacterial tRNA synthetase in an attempt to understand further the evolutionary pathway that has converted a bacterial tRNA synthetase gene into a dual functional yeast gene possessing both cytoplasmic and mitochondrial activities
Summary
We focused on the cross-species and crosscompartmental complementation activities of a bacterial tRNA synthetase in an attempt to understand further the evolutionary pathway that has converted a bacterial tRNA synthetase gene into a dual functional yeast gene possessing both cytoplasmic and mitochondrial activities. The E. coli enzyme, when targeted to mitochondria, could substitute for the mitochondrial activity of VAS1 without the assistance of a cis-acting nonspecific TRBD. These results, together with others, suggest that acquiring a cis- or trans-acting TRBD might be necessary and sufficient for functioning by a yeast cytoplasmic tRNA synthetase, which might explain why so many yeast cytoplasmic aaRSs contain an N- or C-terminal Ad [12]. Obtaining a cis- or trans-acting TRBD does not appear to be necessary for functioning of most yeast mitochondrial aaRSs
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