Leucyl-tRNA Synthetase from the Hyperthermophilic Bacterium Aquifex aeolicus Recognizes Minihelices

Abstract

Aminoacylation of the minihelix mimicking the amino acid acceptor arm of tRNA has been demonstrated in more than 10 aminoacyl-tRNA synthetase systems. Although Escherichia coli or Homo sapiens cytoplasmic leucyl-tRNA synthetase (LeuRS) is unable to charge the cognate minihelix or microhelix, we show here that minihelix Leu is efficiently charged by Aquifex aeolicus synthetase, the only known heterodimeric LeuRS (␣␤-LeuRS). Aminoacylation of minihelices is strongly dependent on the presence of the A73 identity nucleotide and greatly stimulated by destabilization of the first base pair as reported for the E. coli isoleucyl-tRNA synthetase and methionyl-tRNA synthetase systems. In the E. coli LeuRS system, the anticodon of tRNA Leu is not important for recognition by the synthetase. However, the addition of RNA helices that mimic the anticodon domain stimulates minihelix Leu charging by ␣␤-LeuRS, indicating possible domain-domain communication within ␣␤-LeuRS. The leucine-specific domain of ␣␤-LeuRS is responsible for minihelix recognition. To ensure accurate translation of the genetic code, LeuRS functions to hydrolyze misactivated amino acids (pretransfer editing) and misaminoacylated tRNA (posttransfer editing). In contrast to tRNA Leu , minihelix Leu is unable to induce posttransfer editing even upon the addition of the anticodon domain of tRNA. Therefore, the context of tRNA is crucial for the editing of mischarged products. However, the minihelix Leu cannot be misaminoacylated, perhaps because of the tRNA-independent pretransfer editing activity of ␣␤-LeuRS.Aminoacyl-tRNA synthetases (aaRSs) 1 establish the genetic code by catalyzing the esterification of cognate amino acids to their specific transfer RNAs (tRNA) that bear the corresponding anticodons, which are defined by the genetic code (1). Aminoacylation of tRNA catalyzed by aaRSs is a two-step reaction: (a) activation of amino acids with ATP by formation of aminoacyl adenylate and (b) transfer of the aminoacyl moiety from the aminoacyl adenylate to the cognate tRNA substrate. On the basis of the architecture of the conserved active site domain, aaRSs are divided into two groups (2). Class I enzymes have an active site based on the Rossmann fold, whereas the conserved active core of class II enzymes contains an antiparallel ␤-sheet flanked by ␣-helices.One hypothesis about the assembly of aminoacyl-tRNA synthetases is that the enzymes are assembled in a modular fashion, incorporating new domains around the conserved active site representing ancestral aaRS such as the tRNA-binding domain and editing domain (3). Interestingly, two domains of tRNA (specifically the amino acid-accepting stem (minihelix) and anticodon stem biloop (SBL)), which interact with the aminoacylation active site and tRNA-binding domain of aaRS, respectively, may have arisen independently (4). Chargeable minihelices and smaller helices (e.g. microhelices of 7 bp) mimicking the acceptor stem have been identified in more than 10 aaRS systems (5). Thus, whereas it is recognized that aaRS...

Recognition of tRNA Leu by Aquifex aeolicus leucyl-tRNA synthetase during the aminoacylation and editing steps

“…It has a unique αβ heterodimeric structure that mimics primitive aaRS enzymes with separate active sites and tRNA-binding domains (25). Aa LeuRS aminoacylates a cognate minihelix believed to be the tRNA ancestor (26), and its editing domain can function as an isolated domain (27). To investigate the tRNA–synthetase interactions, we performed enzymatic and chemical probing on tRNA Leu complexed with LeuRS as well as kinetic analysis on tRNA Leu variants.…”

A present-day aminoacyl-tRNA synthetase with ancestral editing properties

“…2C). AaLeuRS and EcIleRS efficiently aminoacylated minihelix LIV , compared with minihelix Leu (Xu et al 2004b) and minihelix Ile , respectively (Nordin and Schimmel 1999).…”

“…The first 59-terminal nucleotide was deleted to increase the flexibility of the single-stranded 39 end ( Fig. 2B; Nordin and Schimmel 1999;Xu et al 2004b). Leucine, isoleucine, valine, and threonine were attached to the minihelix LIV by AaLeuRS, EcIleRS, EcValRS, and EcValRS-T 222 P (Döring et al 2001), respectively (Fig.…”

“…RNA from minihelix LIV was obtained by T7 RNA transcription of annealed oligodeoxynucleotides. In vitro transcription and purification of minihelix LIV followed the protocol previously described (Xu et al 2004b). …”

“…A. aeolicus and E. coli tRNA Ile (GAU) were overexpressed in E. coli and purified as previously described (Xu et al 2002). Transcripts from A. aeolicus and E. coli tRNA Leu (GAG) and tRNA Val (UAC) were prepared according to Xu et al (2004b).…”