Importance of the Anticodon Sequence in the Aminoacylation of tRNAs by Methionyl-tRNA Synthetase and by Valyl-tRNA Synthetase in an Archaebacterium

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 (CAU3 CUA and CAU3 GAC) in the anticodon sequence of the initiator tRNA on its recognition by the aminoacyl-tRNA synthetases in vivo. The CAU3 CUA mutant was not aminoacylated to any significant extent in vivo, suggesting the importance of the anticodon in aminoacylation of tRNA by methionyltRNA 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 CAU3 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.The sequence and/or structural determinants in the anticodon and in the acceptor stem of tRNAs play an important role in discrimination among tRNAs by aminoacyl-tRNA synthetases. The crystal structure analysis of several aminoacyl-tRNA synthetase-tRNA complexes from eubacteria and eukarya has provided a substantial amount of information on the molecular details of interactions involving these determinants (1-5). However, little is known either at the biochemical level or at the structural level on interaction between aminoacyl-tRNA synthetases and tRNA in archaea. The results of recent work, 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. First, in contrast to lysyl-tRNA synthetases from eubacteria and eukarya, which in general belong to Class II (5), lysyltRNA synthetase from Methanococcus jannaschii belongs to Class I (6, 7). Second, a single polypeptide of M. jannaschii has the activity (8, 9) of both a cysteinyl-tRNA synthetase and prolyl-tRNA synthetase. Third, the M. jannaschii tyrosyl-tRNA synthetase has a truncated C-terminal region and lacks most of the tRNA anticodon binding region seen in tyro...

Identification and characterization of a tRNA decoding the rare AUA codon in Haloarcula marismortui

“…2). It is known that the stability of the ester link between tRNA and amino acid is determined by the nature of the amino acid attached to the tRNA (Matthaei et al 1966;Drabkin and RajBhandary 1998;Ramesh and RajBhandary 2001), with isoleucine and valine being the most stable, and methionine being much less stable. Aminoacyl-tRNAs present in total RNA were isolated from H. marismortui under acidic conditions to retain the aminoacyl-ester linkage and were subjected to base-catalyzed deacylation (for details, see Materials and Methods).…”

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

“…The determination of deacylation kinetics has been used earlier to characterize the nature of the amino acid attached in vivo to a mutant human initiator tRNA initiating protein synthesis from non-AUG codons [17], and to establish the importance of the anticodon sequence for recognition of tRNAs by MetRS and valyl-tRNA synthetase in archaea [54].…”

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs

“…H. marismortui ATCC 43,049 was kindly provided by Dr. Peter Moore (Department of Chemistry, Yale University). The E. coli strains used in this work, E. coli XL1-blue and E. coli GM2163, have been described before (Ramesh and RajBhandary 2001). General manipulations of H. marismortui, H. volcanii, and E. coli were performed according to standard procedures (DasSarma and Fleischmann 1995;Sambrook and Russell 2001).…”

“…The gene for H. marismortui tRNA 2 Ile was first cloned into the vector pUCsptProM (Ramesh and RajBhandary 2001). For this, the gene for H. marismortui tRNA 2 Ile was PCR amplified from H. marismortui genomic DNA and inserted into the XbaI and BamHI sites of plasmid pUCsptProM downstream from the tRNA Lys promoter to generate pUCHmaIle2WT.…”

“…After confirming the sequences of the desired wild-type and mutant tRNA genes, the respective plasmids were used to transform E. coli GM2163 (dam − ). Plasmid DNA isolated from this strain was then used to transform H. volcanii as described (DasSarma and Fleischmann 1995;Ramesh and RajBhandary 2001). Transformants were grown in H. volcanii medium containing 4 µg/mL mevinolin (US Biologicals).…”