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

Min-Gang Xu,Ming-Wei Zhao,En-Duo Wang

doi:10.1074/jbc.m403018200

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 (alpha beta-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 alpha beta-LeuRS, indicating possible domain-domain communication within alpha beta-LeuRS. The leucine-specific domain of alpha beta-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 alpha beta-LeuRS.

Highlights

Aminoacyl-tRNA synthetases1 establish the genetic code by catalyzing the esterification of cognate amino acids to their specific transfer RNAs that bear the corresponding anticodons, which are defined by the genetic code [1]
We show that in the deep-rooted bacterium A. aeolicus ␣␤-leucyl-tRNA synthetase (LeuRS) consisting of two subunits charges minihelixLeu, suggesting that the ability of the “modern” canonical single peptide LeuRS to recognize the ancient tRNA molecule may have been lost in evolution, whereas ancient ␣␤-LeuRS retains the mechanism to recognize the minihelix
In view of the tRNA-aaRS co-evolution hypothesis, it is noticed that the organization of the aaRS-tRNA complex is consistent with a scheme whereby assembly of the LeuRS subunits accompanies dimerization of the two minihelices, which leads to the full two-domain tRNA structure

Summary

EXPERIMENTAL PROCEDURES

Materials—L-Leucine, L-isoleucine, L-norvaline, 5Ј-GMP, ATP, GTP, CTP, UTP, tetrasodium pyrophosphate, inorganic pyrophosphatase, and dithiothreitol were purchased from Sigma. T7 transcripts were generated in a reaction mixture containing 40 mM Tris-HCl, pH 8.0, 5 mM dithiothreitol, 10 mM MgCl2, 2 mM nucleotide triphosphates, 15 mM 5Ј-GMP, 0.5 unit/ml inorganic pyrophosphatase, and 2 mg/ml pure T7 RNA polymerase. Aminoacylation assays for minihelices were performed in a 70-␮l reaction mixture containing 25 mM Tris-HCl, pH 6.8, 10 mM MgCl2, 1 mM ATP, 1.3 ␮M L-[3H]leucine (1 mCi/ml) or 1.2 ␮M L-[3H]isoleucine (1 mCi/ml), 15 ␮M minihelix, 0.05 unit/ml inorganic pyrophosphatase, and 2 ␮M LeuRS. Assays with minihelices or tRNA were performed at 60 °C in a 70-␮l reaction mixture containing 30 mM Tris-HCl, pH 6.8, 12 mM MgCl2, 5 mM dithiothreitol, 1 unit/␮l ribonuclease inhibitor, 2 units/ml inorganic pyrophosphatase, 2 mM [␥-32P]ATP (50 –70 cpm/ pmol), 15 mM norvaline, 15 ␮M Aa-minihelix or A. aeolicus tRNALeu, and 1 ␮M ␣␤-LeuRS. Background ATP hydrolysis in the absence of RNA was performed for each determination

RESULTS

RNA transcripts kcat

DISCUSSION