Abstract
The mode of recognition of tRNAs by aminoacyl-tRNA synthetases and translation 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 plasmid capable of expressing the tRNA in these cells. Analysis of tRNAs in vivo 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 formylmethionine. We have analyzed the effect of two different mutations (CAU-->CUA and CAU-->GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU-->CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyl-tRNA synthetase. This mutant initiator tRNA can, however, be aminoacylated in vitro by the Escherichia coli glutaminyl-tRNA synthetase, suggesting that the lack of aminoacylation is due to the absence in H. volcanii of a synthetase, which recognizes the mutant tRNA. Archaebacteria lack glutaminyl-tRNA synthetase and utilize a two-step pathway involving glutamyl-tRNA synthetase and glutamine amidotransferase to generate glutaminyl-tRNA. The lack of aminoacylation of the mutant tRNA indicates that this mutant tRNA is not a substrate for the H. volcanii glutamyl-tRNA synthetase. The CAU-->GAC anticodon mutant is most likely aminoacylated with valine in vivo. Thus, the anticodon plays an important role in the recognition of tRNA by at least two of the halobacterial aminoacyl-tRNA synthetases.
Highlights
Spurred by a knowledge of the complete genome sequences of several archaea, have highlighted some interesting and surprising differences between three archaeal aminoacyl-tRNA synthetases and their eubacterial and eukaryal counterparts
We show that the anticodon sequence in the tRNA plays an important role in the recognition of tRNAs by at least two of the aminoacyl-tRNA synthetases, the methionyltRNA synthetase (MetRS)1 and the valyl-tRNA synthetase (ValRS)
The H. volcanii Initiator tRNA—In the absence of an archaeal in vitro protein synthesis system that can translate a natural mRNA or in the absence of any genetic analysis involving components of the translational initiation machinery, identification of the initiator tRNAiMet in halobacteria is based on the ability of the tRNA to be aminoacylated with E. coli MetRS and to be formylated by the E. coli MTF and the ability of the fMet-tRNAiMet to initiate protein synthesis in an E. coli cell-free system [24]
Summary
Spurred by a knowledge of the complete genome sequences of several archaea, have highlighted some interesting and surprising differences between three archaeal aminoacyl-tRNA synthetases and their eubacterial and eukaryal counterparts. This wash eliminated the signal from the probe hybridizing to the wild type initiator tRNAiMet. Measurement of Rates of Deacylation of Aminoacyl-tRNAs—Total tRNA was prepared from H. volcanii/pWL201HvMetiG34C36 under acidic conditions as described above. Based on these results suggesting the stable maintenance of plasmids containing the wild type initiator tRNA gene in H. volcanii, we generated the constructs containing the U35A36 and G34C36 mutations in the anticodon sequence of the initiator tRNA.
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