Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability

Abstract

Editing of misactivated amino acids by class I tRNA synthetases is encoded by a specialized internal domain specific to class I enzymes. In contrast, little is known about editing activities of the structurally distinct class II enzymes. Here we show that the class II alanyl-tRNA synthetase (AlaRS) has a specialized internal domain that appears weakly related to an appended domain of threonyl-tRNA synthetase (ThrRS), but is unrelated to that found in class I enzymes. Editing of misactivated glycine or serine was shown to require a tRNA cofactor. Specific mutations in the aforementioned domain disrupt editing and lead to production of mischarged tRNA. This class-specific editing domain was found to be essential for cell growth, in the presence of elevated concentrations of glycine or serine. In contrast to ThrRS, where the editing domain is not found in all three kingdoms of living organisms, it was incorporated early into AlaRSs and is present throughout evolution. Thus, tRNA-dependent editing by AlaRS may have been critical for making the genetic code sufficiently accurate to generate the tree of life.

Fig. 1. (A) tRNA-stimulated editing by AlaRS at room temperature (22°C) (pH 7.9) in the presence of glycine and serine with and without tRNA^Ala. Inset: fragment 461N and wild-type AlaRS in the presence of glycine or serine and tRNA^Ala. (B) Sequences containing clusters of conserved amino acids in a central region in AlaRS are shown. Numbering is for E.coli AlaRS. A similar sequence of E.coli ThrRS is given below. The relative position in the sequence of the domain in AlaRS versus the analogous domain of ThrRS is depicted above.

Fig. 2. Mischarging of tRNA^Ala at room temperature (22°C) (pH 7.5) by C666A AlaRS and by fragment 461N with glycine (A) or serine (B).

Fig. 3. Editing (ATPase) activity of wild-type and C666A AlaRS at room temperature (22°C) (pH 7.9) in the presence of glycine (A) and serine (B). tRNA-independent hydrolysis of ATP was subtracted as background. (C) Deacylation of Gly-tRNA^Ala or (D) Ser-tRNA^Ala by wild-type and C666A AlaRS and fragment 461N at room temperature (22°C) (pH 7.5).

Fig. 4. Growth of cells containing C666A or wild-type AlaRS. (A) Growth in the presence of a radial gradient of glycine or serine. (B) A 5 µl aliquot of cells bearing either C666A or wild-type AlaRS was streaked onto plates with no amino acid, 2.5 mM serine or 80 mM glycine. All experiments were incubated for 20–24 h at 37°C.

Fig. 5. Phylogenetic analysis of the editing domain of AlaRS. (A) Maximum parsimony tree derived from the alignment of the editing domains of AlaRS (corresponding to G535–S686 of E.coli AlaRS), rooted with AlaXp (Schimmel and Ribas De Pouplana, 2000). The numbers in branch nodes correspond to bootstrap frequencies after 1000 bootstrap cycles in parsimony and distance trees. The root of the tree was derived from the position of the AlaXp clade (shown as a red branch). The overall geometry obtained with the editing domain sequences is identical to that obtained for the active site domain alone (Chihade et al., 2000), indicating that the editing domain has evolved with the active site of the protein from the inception of modern AlaRSs. (B) Distribution of the editing domain throughout the tree of life in ThrRS (red squares) and AlaRS (blue circles).

Fig. 6. (A) AlaRS editing domain model and known E.coli ThrRS structure (coordinates from Sankaranarayanan et al., 1999). (B) Proposed model for discrimination by the editing site of AlaRS. Top: illustration of ValRS discrimination against the hydrophobic methyl group of valine and for the hydroxyl of threonine or a side chain smaller than valine. ‘X’ denotes failure to accommodate the side chain. Bottom: AlaRS chemical discrimination by repulsion of a methyl group or accomodation through hydrogen bonding to the serine hydroxyl. The absence of a side chain allows glycine to be accomm odated.