A freestanding proofreading domain is required for protein synthesis quality control in Archaea

Abstract

Threonyl-tRNA synthetase (ThrRS) participates in protein synthesis quality control by selectively editing the misacylated species Ser-tRNA(Thr). In bacteria and eukaryotes the editing function of ThrRS resides in a highly conserved N-terminal domain distant from the active site. Most archaeal ThrRS proteins are devoid of this editing domain, suggesting evolutionary divergence of quality-control mechanisms. Here we show that archaeal editing of Ser-tRNAThr is catalyzed by a domain unrelated to, and absent from, bacterial and eukaryotic ThrRSs. Despite the lack of sequence homology, the archaeal and bacterial editing domains are both reliant on a pair of essential histidine residues suggestive of a common catalytic mechanism. Whereas the archaeal editing module is most commonly part of full-length ThrRS, several crenarchaeal species contain individual genes encoding the catalytic (ThrRS-cat) and editing domains (ThrRS-ed). Sulfolobus solfataricus ThrRS-cat was shown to synthesize both Thr-tRNAThr and Ser-tRNAThr and to lack editing activity against Ser-tRNAThr. In contrast, ThrRS-ed lacks aminoacylation activity but can act as an autonomous protein in trans to hydrolyze specifically Ser-tRNAThr, or it can be fused to ThrRS-cat to provide the same function in cis. Deletion analyses indicate that ThrRS-ed is dispensable for growth of S. solfataricus under standard conditions but is required for normal growth in media with elevated serine levels. The growth phenotype of the ThrRS-ed deletion strain suggests that retention of the discontinuous ThrRS quaternary structure relates to specific physiological requirements still evident in certain Archaea.

Fig. 1.

Disruption of the SSO0384 locus. (A) Outline of the SSO0384 disruption strategy. The locations of PCR primers are indicated by arrows. The AvrII site of SSO0384 is located at nucleotide 524. LacS is positioned in a reverse orientation relative to SSO0384. (B) PCR analysis of the disrupted and WT SSO0384 locus: lane 1, 1-kb DNA ladder; lane 2, amplification of SSO0384 from disruption mutant by using primers SSO0384-HindIII-F and SSO0384-HindIII-R; lane 3, same as lane 2 but digested with HindIII; lane 4, SSO0384 amplified from WT; lane 5, same as lane 4 but digested with HindIII; lane 6, lacS amplified from WT by using primers lacS-AvrII-F and lacS-AvrII-R.

Fig. 2.

Schematic diagram of the domain structure of various ThrRSs and detailed alignment around the region that interacts with the zinc atom. Highly conserved residues are highlighted in blue, and residues involved in binding of zinc are in red. Numbering corresponds to the residue immediately to the right. Abbreviations are as follows: EC, E. coli; BS, Bacillus subtilis; MJ, M. jannaschii; PA, P. aerophilum; SS1, S. solfataricus archaeal-type ThrRS-ed; SS2, S. solfataricus bacterial-type ThrRS-cat.

Fig. 3.

Mischarging and proofreading of tRNA^Thr. (A) Aminoacylation of M. jannaschii tRNA^Thr (2 μM) with 300 μM [³H]serine by ThrRSs: ○, 1 μM ThrRS-cat; •, 1 μM ThrRS-cat plus 1 μM ThrRS-ed; □, 0.2 μM MJThrRS. (Inset) Aminoacylation of M. jannaschii tRNA^Thr (2 μM) with 50 μM[³H]threonine by ThrRSs: ○, 1 μM ThrRS-cat; •, 1 μM ThrRS-cat plus 1 μM ThrRS-ed; □, 0.2 μM MJThrRS. (B) Deacylation of Ser-tRNA^Thr by various ThrRSs: □, 30 nM M. jannaschii ThrRS; •, 30 nM S. solfataricus ThrRS-ed; ▴, 200 nM E. coli ThrRS; ○, 200 nM S. solfataricus ThrRS-cat; ▪, no enzyme.

Fig. 4.

Multiple sequence alignments of the editing domains in archaeal-type ThrRSs inferred by clustalx (30). Residues analyzed by alanine-scanning mutagenesis of M. jannaschii ThrRS are indicated with asterisks. Abbreviations are as follows: A.per, A. pernix (NP_146977); S.sol, S. solfataricus (NP_341922); S.tok, S. tokodaii (NP_378184); M.jan, M. jannaschii (NP_248192); M.the, Methanothermobacter thermautotrophicus (NP_276569); P.hor, Pyrococcus horikoshii (NP_142646); A.ful, Archaeoglobus fulgidus (NP_069384); P.aer, P. aerophilum (NP_559516) (GenBank accession numbers are in parentheses).

Fig. 5.

Site-directed mutagenesis of the archaeal-type editing domain. (A) Editing activity in M. jannaschii ThrRS mutants. Shown are bar graphs comparing the posttransfer editing activity of Ser-tRNA^Thr by M. jannaschii ThrRS mutants. Results are based on the initial rate of deacylation with WT ThrRS activity set at 100%. (B) Aminoacylation of M. jannaschii tRNA^Thr (2 μM) with [³H]serine (300 μM) in the presence of ThrRSs: •, 0.4 μM H84A mutant; ○, 0.4 μM H131A; ▴, 0.4 μM C128A.

Fig. 6.

Aminoacylation and editing activities of the chimeric S. solfataricus ThrRS-ed/ThrRS-cat. (A) Aminoacylation of M. jannaschii tRNA^Thr (2 μM) with 50 μM [³H]threonine: •, 1 μM SSThrRS-cat; ○, 1 μM ThrRS-ed/ThrRS-cat. (B) Deacylation of Ser-tRNA^Thr (≈0.5 μM): •, 10 nM ThrRS-ed; ○, 10 nM ThrRS-ed/ThrRS-cat; ▪, no enzyme.

Fig. 7.

Effect of serine and threonine on growth of the S. solfataricus WT (PBL2025) and the ThrRS-ed deletion (PLB2034) strain in minimal medium. □, PBL2025, no amino acid addition; ○, PBL2025 plus 0.5 mM Ser; ▿, PBL2025 plus 0.5 mM Ser+Thr; ▪, PBL2034, no amino acid addition; •, PBL2034 plus 0.5 mM Ser; ▴, PBL2034 plus 0.5 mM Ser+Thr.