Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

Abstract

Mycoplasma leucyl-tRNA synthetases (LeuRSs) have been identified in which the connective polypeptide 1 (CP1) amino acid editing domain that clears mischarged tRNAs are missing (Mycoplasma mobile) or highly degenerate (Mycoplasma synoviae). Thus, these enzymes rely on a clearance pathway called pretransfer editing, which hydrolyzes misactivated aminoacyl-adenylate intermediate via a nebulous mechanism that has been controversial for decades. Even as the sole fidelity pathway for clearing amino acid selection errors in the pathogenic M. mobile, pretransfer editing is not robust enough to completely block mischarging of tRNA(Leu), resulting in codon ambiguity and statistical proteins. A high-resolution X-ray crystal structure shows that M. mobile LeuRS structurally overlaps with other LeuRS cores. However, when CP1 domains from different aminoacyl-tRNA synthetases and origins were fused to this common LeuRS core, surprisingly, pretransfer editing was enhanced. It is hypothesized that the CP1 domain evolved as a molecular rheostat to balance multiple functions. These include distal control of specificity and enzyme activity in the ancient canonical core, as well as providing a separate hydrolytic active site for clearing mischarged tRNA.

Fig. 1.

M. synoviae LeuRS mischarges tRNA^Leu. (A) Leucylation by M. synoviae LeuRS was carried out by using 21 µM [¹⁴C]leucine (50 μCi/mL), 4 µM tRNA^Leu, and 100 nM enzyme. (B) Isoleucine mischarging by M. synoviae LeuRS incorporated 40 µM [³H]isoleucine (166 μCi/mL), 4 µM tRNA^Leu, and 1 µM enzyme. M. mobile LeuRS was used as a positive control. (C) Deacylation reactions for M. synoviae LeuRS contained ∼6.5 µM [³H]Ile-tRNA^Leu and 100 nM enzyme. E. coli LeuRS was used as the positive control. Abbreviations are as follows: ■, M. synoviae LeuRS (Ms); ▲, M. mobile LeuRS (Mm); ▼, E. coli LeuRS (Ec); and ◆, no enzyme control (noE). Error bars represented SDs calculated from triplicated reactions.

Fig. 2.

M. mobile LeuRS exhibits pretransfer editing activity. Reaction mixtures contained 10 μM M. mobile tRNA^Leu or a tRNA analog with 2′-deoxyadenosine substituted for the A₇₆ nucleotide (2′dA-tRNA) and 1 μM M. mobile LeuRS. Amino acid-dependent AMP formation is measured by TLC of reaction aliquots from ATPase reactions with 5 mM isoleucine (A) or 5 mM leucine (B). Aminoacyl-adenylate was measured with 5 mM isoleucine (C) or 5 mM leucine (D). Fractions indicated by the y axis represent the intensity of the spot representing [³²P]adenylate divided by the total intensity of ³²P in the lane. Abbreviations are as follows: ■, isoleucine only (Ile); ▲, isoleucine and tRNA (Ile+tRNA); ▼, isoleucine and 2′dA-tRNA (Ile+2'dA); ◆, leucine only (Leu); ●, leucine and tRNA (Leu+tRNA); □, leucine and 2′dA-tRNA (Leu+2'dA); and ×, no amino acid control (No AA). Error bars represent SDs derived from triplicated reactions.

Fig. 3.

X-ray crystal structure of M. mobile LeuRS Leu-AMS complex. (A) A ribbon diagram of the protomer with electron density of C-terminal domain is colored as follows: catalytic domain, yellow; four-helix bundle domain, red; C-terminal domain, pink; Zn binding domain, purple; KMSKS loop, blue; two linking β-strands, cyan. The Leu-AMP analog, Leu-AMS, is shown in stick model. A color-coordinated cartoon of the primary sequence is shown below the structure. (B) The 2F_o-F_c electron density map of the aminoacylation active site is contoured at 1.0 σ (black mesh). The Leu-AMS is highlighted in green, and interacting amino acid residues are labeled and shown in gray.

Fig. 4.

Aminoacylation and misaminoacylation activities of M. mobile LeuRSs that contain hybrid CP1 domains. Aminoacylation of 4 μM tRNA^Leu in presence of (A) 21 μM [³H]leucine (318 μCi/mL), (B) 21 μM [³H]isoleucine (166 μCi/mL), (C) 20.2 μM [³⁵S]methionine (115 μCi/mL), and (D) 20 μM [¹⁴C]valine (259 μCi/mL). Symbols used are as follows: ■, MmLeuRS (Mm); ▲, MmLeuRS/CP1^Leu (Mm/CP1^Leu); ▼, MmLeuRS/CP1^Ile (Mm/CP1^Ile); and ◆, MmLeuRS/CP1^Val (Mm/CP1^Val). Error bars represent SDs from triplicated reactions.

Fig. 5.

CP1 addition to hybrid M. mobile LeuRS enhances pretransfer editing. Reaction mixture contained 1 μM enzyme, 10 μM tRNA^Leu, or tRNA^Leu with A₇₆ replaced by dideoxyadenosine (ddA), 18.1 μM [α-³²P]ATP (40 μCi/mL), and 2.5 mM isoleucine. The AMP formation activities are measured for Mm LeuRS (A), MmLeuRS/CP1^Leu (Mm/ CP1^Leu) (B), MmLeuRS/CP1^Ile (Mm/CP1^Ile) (C), and MmLeuRS/CP1^Val (Mm/CP1^Val) (D). Symbols used are as follows: ◆, reaction without amino acid present (no AA); ▲, isoleucine (Ile); ▼, isoleucine with tRNA (Ile+tRNA); and ■, isoleucine with ddtRNA (Ile+ddA). Error bars represent the SD values based on three separate experiments.

Fig. 6.

M. synoviae LeuRS CP1 addition to M. mobile LeuRS suppresses mischarging via enhanced pretransfer editing activity. (A) Aminoacylation and misaminoacylation activity of Mm/Ms LeuRS was carried out with 4 μM tRNA^Leu in the presence of 21 μM [³H]leucine (318 μCi/mL) with 1 μM enzyme or 21 μM [³H]isoleucine (97 μCi/mL) with 1 μM enzyme. Symbols used are as follows: □, Leu-tRNA^Leu and △, Ile-tRNA^Leu. (B) The AMP formation activity was measured for Mm/Ms LeuRS using 1 μM enzyme, 10 μM tRNA^Leu, or tRNA^Leu with A₇₆ replaced by dideoxyadenosine (ddA-tRNA), 18.1 μM [α-³²P]ATP, and 2.5 mM isoleucine. Symbols used are as follows: ◆, reaction without amino acid present (no AA); ▲, isoleucine (Ile); ▼, isoleucine with tRNA (Ile+tRNA); and ■, isoleucine with ddA-tRNA (Ile+ddA). Error bars represent the SD values based on three separate experiments.