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

Abstract

Recognition of tRNA by the cognate aminoacyl-tRNA synthetase during translation is crucial to ensure the correct expression of the genetic code. To understand tRNA(Leu) recognition sets and their evolution, the recognition of tRNA(Leu) by the leucyl-tRNA synthetase (LeuRS) from the primitive hyperthermophilic bacterium Aquifex aeolicus was studied by RNA probing and mutagenesis. The results show that the base A73; the core structure of tRNA formed by the tertiary interactions U8-A14, G18-U55 and G19-C56; and the orientation of the variable arm are critical elements for tRNA(Leu) aminoacylation. Although dispensable for aminoacylation, the anticodon arm carries discrete editing determinants that are required for stabilizing the conformation of the post-transfer editing state and for promoting translocation of the tRNA acceptor arm from the synthetic to the editing site.

Figure 1.

Nuclease probing of AatRNA^Leu_GAG in the free form or in complex with AaLeuRS. tRNA^Leu_GAG was labeled at its 5′-end (A) or 3′-end (B). Probing was conducted in the presence (+) or absence (−) of LeuRS. OH and T1 are ladders of the tRNA under the denaturing condition; Ctrl is the control without any probe. The probes comprised RNase T1, RNase T2 and RNase V1. Numbers refer to tRNA nucleotide positions. (C) Cloverleaf structure of tRNA summarizing the reactivity changes observed in the tRNA following AaLeuRS binding. The symbols and color codes for the probes are indicated in the figure. Three intensities of cuts/modifications for each probe are shown (strong, medium and moderate).

Figure 2.

Nuclease probing of AatRNA^Leu_CAA in the free form or in complex with AaLeuRS. (A) tRNA^Leu_CAA was labeled at its 5′-end or 3′-end. Probing was done in the presence (+) or absence (−) of LeuRS. OH and T1 are ladders of the tRNA under the denaturing condition; Ctrl is the control without any probe. The probes comprised RNase T1, RNase T2 and RNase V1. Numbers refer to tRNA nucleotide positions. (B) Cloverleaf structure of tRNA summarizing the reactivity changes observed in the tRNA following AaLeuRS binding. The symbols and color codes for the probes are indicated in the figure. Three intensities of cuts/modifications for each probe are shown (strong, medium and moderate).

Figure 3.

Iodine probing of AatRNA^Leu_GAG containing phosphorothioate nucleotides. (A) AatRNA^Leu_GAG was labeled at its 5′-end and probed in the presence (+) or absence (−) of AaLeuRS; Ctrl is the control without iodine. (B) Cloverleaf structure of AatRNA summarizing the protections observed in the tRNA following AaLeuRS binding. Three intensities of protections are shown. (C) View of the T. thermophilus LeuRS/tRNA^Leu complex (2BYT) (10). The enzyme is shown in red and the tRNA is in blue. The tRNA nucleotides protected from iodine cleavage in AatRNA are highlighted in yellow.

Figure 4.

Summary of AatRNA^Leu constructs in this study. The AatRNA variants are derived from AatRNA^Leu_GAG. The arrows indicate the mutation locations; bp, base pair; Δ, deletions. Colors are used for the multiple mutants, and the asterisks indicate the double mutation 19–56.

Figure 5.

Effect of AatRNA^Leu_GAG mutations on the editing reactions of AaLeuRS. In (A)–(C), different colors represent various AatRNA^Leu_GAG variants: AatRNA^Leu_GAG (wild-type), black; AatRNA^Leu_GAG (A35U), red; AatRNA^Leu_GAG (A35C), green; AatRNA^Leu_GAG (A35G), yellow; AatRNA^Leu_GAG (ΔAC), blue; and AatRNA^Leu_GAG (C41U), pink. Cyan curve shows the background rate in the ATP consumption assay in the absence of isoleucine in (A) or the spontaneous hydrolysis in the absence of the enzyme in (B). (A) ATP consumption during total editing by AaLeuRS in the presence of 50 mM isoleucine and 5 μM AatRNA^Leu mutant transcripts. (B) Hydrolysis of Ile-tRNA^Leu_GAG for AatRNA^Leu_GAG mutants by 5 nM AaLeuRS. The spontaneous hydrolysis in the absence of the enzyme (<10%) were subtracted. (C) Isoleucylation rates of 5 μM AatRNA^Leu mutants by 1 μM LeuRS. Data are averaged from three independent assays.