Importance of the anticodon sequence in the aminoacylation of tRNAs by methionyl-tRNA synthetase and by Valyl-tRNA synthetase in an archaebacterium

The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translat ion factors is largely unknown in archaebacteria. To study this process, we have cloned the wild type initiator tRNA gene from the moderate halophilic archaebacterium Haloferax volcanii and mutants derived from it into a plas mid capable of expressing the tRNA in these cells. Analysis of tRNAs in viv o show that the initiator tRNA is aminoacylated but is not formylated in H. volcanii, This result provides direct support for the notion that protein synthesis in archaebacteria is initiated with methionine and not with formy lmethionine. We have analyzed the effect of two different mutations (CAU--> CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its r ecognition by the aminoacyl-tRNA synthetases in vivo. The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the imp ortance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthe tase. This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coil glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H. volcanii of a synthetase, whi ch recognizes the mutant tRNA Archaebacteria lack glutaminyl-tRNA synthetas e and utilize a two-step pathway involving glutamyl-tRNA synthetase and glu tamine amidotransferase to generate glutaminyl-tRNA The lack of aminoacylat ion of the mutant tRNA indicates that this mutant tRNA is not a substrate f or the H, volcanii glutamyl-tRNA synthetase. The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo. Thus, the anticodon play s an important role in the recognition of tRNA by at least two of the halob acterial aminoacyl-tRNA synthetases.