Translational Quality Control by Bacterial Threonyl-tRNA Synthetases

Abstract

Translational fidelity mediated by aminoacyl-tRNA synthetases ensures the generation of the correct aminoacyl-tRNAs, which is critical for most species. Threonyl-tRNA synthetase (ThrRS) contains multiple domains, including an N2 editing domain. Of the ThrRS domains, N1 is the last to be assigned a function. Here, we found that ThrRSs from Mycoplasma species exhibit differences in their domain composition and editing active sites compared with the canonical ThrRSs. The Mycoplasma mobile ThrRS, the first example of a ThrRS naturally lacking the N1 domain, displays efficient post-transfer editing activity. In contrast, the Mycoplasma capricolum ThrRS, which harbors an N1 domain and a degenerate N2 domain, is editing-defective. Only editing-capable ThrRSs were able to support the growth of a yeast thrS deletion strain (ScΔthrS), thus suggesting that ScΔthrS is an excellent tool for studying the in vivo editing of introduced bacterial ThrRSs. On the basis of the presence or absence of an N1 domain, we further revealed the crucial importance of the only absolutely conserved residue within the N1 domain in regulating editing by mediating an N1-N2 domain interaction in Escherichia coli ThrRS. Our results reveal the translational quality control of various ThrRSs and the role of the N1 domain in translational fidelity.

Keywords: aminoacyl tRNA synthetase; protein synthesis; transfer RNA (tRNA); translation; translation control.

FIGURE 1.

Domain structures and primary sequence alignment of the editing active sites of various ThrRSs and the purification of various ThrRS mutants. A, schematic showing the domain compositions of various ThrRSs. The degeneration of the editing domain of McThrRS is indicated by asterisks. The N-terminal domain of MhThrRS is indicated by an ellipse. B, sequence alignment of the editing active sites of the various native ThrRSs. The identified editing active site residues of EcThrRS (bold and light green) are indicated by asterisks and numbered (EcThrRS numbering), and the counterparts of McThrRS (bold and red) have been changed. The editing active site residues of MmThrRS are also indicated (bold and yellow). Ec, E. coli; Mc, M. capricolum; Mm, M. mobile; Aa, Aquifex aeolicus; Tt, Thermus thermophiles; Sa, Staphylococcus aureus. C, approximately 5 μg of each protein was loaded onto a 10% SDS-PAGE gel, as indicated. Standard molecular weights are shown on the left.

FIGURE 2.

Editing capacity of MmThrRS and McThrRS. A, post-transfer editing of Ser-tRNA^Thr by MmThrRS (□), McThrRS (▵), EcThrRS (▾), and MmThrRS-H9A/H13A (▿). Spontaneous hydrolysis of Ser-tRNA^Thr was performed as a control (●). B, a representative TLC image showing the mischarging of [³²P]tRNA^Thr with noncognate Ser. Free [³²P]tRNA^Thr and mischarged [³²P]tRNA^Thr are represented by [³²P]AMP and Ser-[³²P]AMP, respectively. C, quantitative analysis of Ser-[³²P]tRNA^Thr generated by MmThrRS (●), McThrRS (■), and EcThrRS (♦) (as an editing-capable control). The data in A and C represent averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols.

FIGURE 3.

MmThrRS and McThrRS exhibit no tRNA-dependent pretransfer editing activity. A and B, generation of [³²P]AMP in the absence (−tRNA) or presence (+tRNA) of tRNA^Thr by MmThrRS (A) and McThrRS (B), after incubations of 2, 4, 6, 8, and 10 min. A 2-fold dilution of [α-³²P]ATP (initial concentration, 3 mm) was included for quantification. C and D, quantification of AMP formation by MmThrRS (C) and McThrRS (D) with or without tRNA. The data in C and D represent averages of three independent experiments and the corresponding standard errors.

FIGURE 4.

AMP formation of MmThrRS or McThrRS with Thr or Ser. A and B, Thr or Ser-included AMP formation of MmThrRS (A) or McThrRS (B) in the absence of tRNA^Thr. aa-AMP, aminoacyl-adenylate. C, quantification of AMP formation by McThrRS with Thr (○) or Ser (○). Those of MmThrRS with Thr and Ser were not plotted because the former was too low to be accurately calculated. A 2-fold dilution of [α-³²P]ATP (initial concentration, 3 mm) was included for quantification. The data represent averages of three independent experiments and the corresponding standard errors.

FIGURE 5.

Editing-defective ThrRSs are unable to rescue ScThrRS loss of function yeast. A, complementation of an ScThrRS loss of function mutant strain by MmThrRS, MmThrRS-H9A/H13A, and McThrRS. ScThrRS and p425TEF are used as positive and negative controls, respectively. B, aminoacylation of yeast tRNA^Thr isoacceptors by MmThrRS or McThrRS. C, schematic showing the domain compositions of N1 and N2 for the EcThrRS, MmThrRS, McThrRS enzymes, and the various mutants with domain deletions, swaps, or mutations. The aminoacylation and CTDs are shown as the “main body” for clarity. The capacity of the proteins to support the growth of ScΔthrS (complementation) or post-transfer editing is indicated with + and − symbols. ND represents not determined because we were unable to obtain soluble proteins. Two asterisks in the N2 domain indicate degeneracy (in McThrRS) or mutation (in MmThrRS) of the active sites. D, post-transfer editing of the Ser-tRNA^Thr by the McThrRS-MmN2-ΔN1 (■) and MmThrRS-N2M (□). E, rescue of the ScThrRS loss-of-function strain by McThrRS, McThrRS-MmN2 and McThrRS-MmN2-ΔN1. EcThrRS and p425TEF are shown as positive and negative controls, respectively. F, rescue of the ScThrRS loss of function strain by MmThrRS-N2M. MmThrRS and p425TEF are shown as positive and negative controls, respectively. G, steady-state protein levels of MmThrRS, MmThrRS-H9A/H13A, and MmThrRS-N2M, each of which had a His₆ tag at the C terminus. ScThrRS expressed from a rescue plasmid had no His₆ tag at the C terminus. GAPDH was used as the loading control. The data shown in B and D represent averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols.

FIGURE 6.

Addition of the N1 domain of McThrRS is detrimental for MmThrRS function. A, yeast complementation assay with different ThrRSs and MmThrRS-▿McN1. B, steady-state protein levels of MmThrRS, MmThrRS-▿McN1, and ScThrRS. GAPDH is included as a loading control. A nonspecific band is indicated with an asterisk. C, MmThrRS, MmThrRS-▿McN1, and ScThrRS relative protein levels after calculation of the normalized ratio of ThrRSs to GAPDH. The data in C represent averages of three independent experiments and the corresponding standard errors. D, yeast complementation by McThrRS-MmN2 and McThrRS-ΔN1.

FIGURE 7.

Role of N1 domain of EcThrRS. A, k_d determinations for EcThrRS (●) and EcThrRS-ΔN1 (■) for tRNA^Thr. B, yeast complementation by EcThrRS and EcThrRS-ΔN1. ScThrRS and p425TEF were used as positive and negative controls, respectively. C, post-transfer editing of Ser-tRNA^Thr by EcThrRS (■) and EcThrRS-ΔN1 (□). The spontaneous hydrolysis of Ser-tRNA^Thr (●) is included as a negative control. D–G, generation of [³²P]AMP in the absence (−tRNA) or presence (+tRNA) of tRNA^Thr by EcThrRS (D and E) and EcThrRS-ΔN1 (F and G), after incubations of 2, 4, 6, 8, and 10 min. A 2-fold dilution of [α-³²P]ATP (initial concentration, 3 mm) was included for quantification. F and G, quantification of AMP formation by EcThrRS (F) and EcThrRS-ΔN1 (G) with or without tRNA. The data in A, C, E, and G represent averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbo7ls.

FIGURE 8.

The Asp⁴⁶ of the N1 domain controls editing and cell viability. A, sequence alignment of the N1 domains of ThrRSs from various species. Ec, E. coli; Mc, M. capricolum; Sa, S. aureus; Aa, A. aeolicus; Tt, T. thermophiles; Sc, S. cerevisiae; Hs, Homo sapiens. B, crystal structure of the EcThrRS-tRNA^Thr complex (Protein Data Bank code 1QF6) (16) showing the spatial location of the Asp⁴⁶-containing N1 domain and the Tyr¹⁷³- and His¹⁸⁶-containing N2 domain. Protein domains are labeled and shown in different colors, with tRNA in orange. C, yeast complementation results for the various Asp⁴⁶ mutants. D, aminoacylation of tRNA^Thr by the native EcThrRS (●), EcThrRS-D46R (□), and EcThrRS-D46E (♢). E, post-transfer editing of Ser-tRNA^Thr by EcThrRS (●), EcThrRS-D46R (□), and EcThrRS-D46E (♢). The spontaneous hydrolysis of Ser-tRNA^Thr (♦) was included as a negative control. The data in D and E represent averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols.

FIGURE 9.

Recovery of editing activity and yeast complementation. A, spatial localization of Asp⁴⁶, Lys¹³⁶, Tyr¹⁷³, and His¹⁸⁶. The side chains of the Asp⁴⁶, Lys¹³⁶, and Tyr¹⁷³ residues and the main chain of the His¹⁸⁶ residue are shown as sticks (left) and spheres (right). The distance between Asp⁴⁶ and the other residues is indicated. B, yeast complementation by mutants of the Lys¹³⁶ and Tyr¹⁷³ residues. C, the aminoacylation activity of the EcThrRS (●), EcThrRS-Y173R (□), EcThrRS-Y173D (▵), EcThrRS-D46E/Y173F (▴), and EcThrRS-D46E/H186G (■) variants. D, post-transfer editing of Ser-tRNA^Thr by EcThrRS-Y173R (□) and EcThrRS-Y173D (▵). Spontaneous hydrolysis (○) and post-transfer editing of Ser-tRNA^Thr by EcThrRS (■) are included as negative and positive controls, respectively. E, yeast complementation by EcThrRS-D46E, EcThrRS-Y173F, EcThrRS-D46E/Y173F, and EcThrRS-D46E/H186G. F, post-transfer editing of Ser-tRNA^Thr by EcThrRS-D46E/Y173F (▴) and EcThrRS-D46E/H186G (▾). Spontaneous hydrolysis (●) and post-transfer editing of Ser-tRNA^Thr by EcThrRS (■) are included as negative and positive controls, respectively. The data in C, D, and F represent the averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols.