Restoring species-specific posttransfer editing activity to a synthetase with a defunct editing domain.
Aminoacyl-tRNA synthetases are multidomain proteins responsible for the attachment of specific amino acids to their tRNA substrates. Prolyl-tRNA synthetases (ProRSs) are notable due to their particularly diverse architectures through evolution. For example, Saccharomyces cerevisiae ProRS possesses an N-terminal extension with weak homology to a bacterial-specific domain typically present as an insertion (INS) within the aminoacylation active site. The INS domain has been shown to contain a "posttransfer" editing active site responsible for cleaving the aminoacyl-ester bond of misacylated Ala-tRNA(Pro) species. However, wild-type S. cerevisiae ProRS does not perform posttransfer editing in vitro. Here, we show that replacement of the N-terminal domain of S. cerevisiae ProRS with the Escherichia coli INS domain confers posttransfer editing function to this chimeric enzyme, with specificity for yeast Ala-tRNA(Pro). In contrast, the isolated INS domain displays only weak editing activity and lacks tRNA sequence specificity. These results emphasize the modular nature of synthetase editing active sites and demonstrate how in evolution, a weak editing activity can be converted to a more robust state through fusion to the body of a synthetase. In this manner, a single editing module can be distributed to different synthetases, and simultaneously acquire specificity and enhanced activity.
SternJohn
Julius
J
Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Hati
Sanchita
S
Siliciano
Paul G
PG
Musier-Forsyth
Karin
K
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D011392:Proline; D014176:Protein Biosynthesis; D017393:RNA Editing; D012346:RNA, Transfer, Amino Acyl; D029701:Saccharomyces cerevisiae Proteins; D013045:Species Specificity; D013379:Substrate Specificity
D012346:RNA, Transfer, Amino Acyl; D029701:Saccharomyces cerevisiae Proteins; D011392:Proline; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase