Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Abstract

Class I aminoacyl-tRNA synthetases (aaRSs) use a Rossmann-fold domain to catalyze the synthesis of aminoacyl-tRNAs required for decoding genetic information. While the Rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among tRNA substrates, raising the question of how the conserved protein fold adapts to RNA sequence variations. Of interest is the existence of an unpaired C-A mismatch at the 1-72 position unique to bacterial initiator tRNA(fMet) and absent from elongator tRNAs. Here we show that the class I methionyl-tRNA synthetase (MetRS) of Escherichia coli and its close structural homolog cysteinyl-tRNA synthetase (CysRS) display distinct patterns of recognition of the 1-72 base pair. While the structural homology of the two enzymes in the Rossmann-fold domain is manifested in a common burst feature of aminoacylation kinetics, CysRS discriminates against unpaired 1-72, whereas MetRS lacks such discrimination. A structure-based alignment of the Rossmann fold identifies the insertion of an α-helical motif, specific to CysRS but absent from MetRS, which docks on 1-72 and may discriminate against mismatches. Indeed, substitutions of the CysRS helical motif abolish the discrimination against unpaired 1-72. Additional structural alignments reveal that with the exception of MetRS, class I tRNA synthetases contain a structural motif that docks on 1-72. This work demonstrates that by flexible insertion of a structural motif to dock on 1-72, the catalytic domain of class I tRNA synthetases can acquire structural plasticity to adapt to changes at the end of the tRNA acceptor stem.

FIGURE 1.

Sequences and cloverleaf structures of tRNA. (A) EctRNA^fMet, (B) EctRNA^Met, and (C) EctRNA^Cys. The 1-72 base pair of the tRNA acceptor stem is boxed, and substitutions of the base pair are indicated by arrows.

FIGURE 2.

Pre-steady-state aminoacylation of EcMetRS. (A) A representative set of data from the average of two experiments of aminoacylation over time with 1 μM EcMetRS and 10 μM EctRNA^fMet. (B) A representative set of data from the average of two experiments of aminoacylation over time with 1 μM EcMetRS and 10 μM EctRNA^Met. Rate constants k_chem and k_cat are shown in each time course. Errors: SDs.

FIGURE 3.

Effect of 1-72 base pair mutations on aminoacylation. Analysis of the loss of k_chem/K_d in logarithmic scale by substitutions of 1-72 in EctRNA^fMet, EctRNA^Met, and EctRNA^Cys.

FIGURE 4.

Acceptor stem recognition by EcMetRS and EcCysRS. (A) The tRNA-bound structure of A. aeolicus MetRS, showing the CP1 domain of MetRS in green, the Rossmann fold in light green, and tRNA in orange (Protein Data Bank [PDB] identification 2CT8). (B) The tRNA-bound structure of EcCysRS, showing the CP1 domain in magenta, the Rossmann fold in light pink, and tRNA in orange (PDB identification 1U0B). (C) Superimposition of the tRNA-bound EcCysRS and EcMetRS by aligning the 5′ half of tRNA backbones (nucleotides 1–34), revealing the presence of the inserted α-helical motif in EcCysRS (circled in black). (D) Interaction between the α-helical motif in EcCysRS and the G1-C72 nucleotides in EctRNA^Cys. The helical motif is shown in light green, while the tRNA acceptor end is shown in light orange in the background. The residues Q158, Q162, and G166 are highlighted in sticks with carbons in green, while G1 and C72 are shown in sticks with carbons in cyan. (E) Effect of the triple mutations (Q158A, Q162A, and G166A) in EcCysRS on aminoacylation of tRNA. The activity is shown in logarithmic scale, while the 1-72 base pairs of the tRNA are indicated at the bottom of the bar graph. (WT) Wild-type EcCysRS; (TM) triple mutant harboring the Q158A, Q162A, and G166A mutations. Graphs showing the structures are generated by PyMol (Delano Scientific).

FIGURE 5.

Acceptor stem recognition by class I aaRSs. (A) Thermus thermophilus TyrRS (Protein Data Bank [PDB] identification 1H3E); (B) human HsTrpRS (PDB identification 2AZX); (C) T. thermophilus GluRS (PDB identification 1N77); (D) P. horikoshii LeuRS (PDB identification 1WZ2); (E) P. horikoshii ArgRS (PDB identification 2ZUE); and (F) E. coli GlnRS (PDB identification 1O0B). (Cyan) Rossmann fold; (light pink) CP1 domain; (magenta) motif in the CP1 domain that docks on 1-72.

FIGURE 6.

Recognition of 1-72 in tRNA by class I aaRSs. (A) Structure of A. aeolicus MetRS (PDB 2CT8); (B) structure of E. coli CysRS (PDB 1U0B); (C) structure of T. thermophilus GluRS (PDB 1N77); and (D) structure of P. horikoshii LeuRS (PDB 1WZ2).