The RNA acetyltransferase driven by ATP hydrolysis synthesizes N4-acetylcytidine of tRNA anticodon

Abstract

The wobble base of Escherichia coli elongator tRNA(Met) is modified to N(4)-acetylcytidine (ac(4)C), which is thought to ensure the precise recognition of the AUG codon by preventing misreading of near-cognate AUA codon. By employing genome-wide screen of uncharacterized genes in Escherichia coli ('ribonucleome analysis'), we found the ypfI gene, which we named tmcA (tRNA(Met) cytidine acetyltransferase), to be responsible for ac(4)C formation. TmcA is an enzyme that contains a Walker-type ATPase domain in its N-terminal region and an N-acetyltransferase domain in its C-terminal region. Recombinant TmcA specifically acetylated the wobble base of E. coli elongator tRNA(Met) by utilizing acetyl-coenzyme A (CoA) and ATP (or GTP). ATP/GTP hydrolysis by TmcA is stimulated in the presence of acetyl-CoA and tRNA(Met). A mutation study revealed that E. coli TmcA strictly discriminates elongator tRNA(Met) from the structurally similar tRNA(Ile) by mainly recognizing the C27-G43 pair in the anticodon stem. Our findings reveal an elaborate mechanism embedded in tRNA(Met) and tRNA(Ile) for the accurate decoding of AUA/AUG codons on the basis of the recognition of wobble bases by the respective RNA-modifying enzymes.

Figure 1

Chemical structure of N⁴-acetylcytidine (ac⁴C) and secondary structure of E. coli elongator tRNA^Met. (A) Chemical structure of ac⁴C. (B) Secondary structure of E. coli elongator tRNA^Met with modified nucleosides: 4-thiouridine (s⁴U), 2′-O-methylguanosine (Gm), dihydrouridine (D), N⁴-acetylcytidine (ac⁴C), N⁶-threonylcarbamoyladenosine (t⁶A), pseudouridine (Ψ), 7-methylguanosine (m⁷G), 3-(3-amino-3-carboxypropyl) uridine (acp³U) and 5-methyluridine (m⁵U).

Figure 2

Mass spectrometric analysis of total nucleosides from the ΔypfI strain and growth phenotype of the ΔypfI/ΔdusC strain. (A) LC/MS analysis of total nucleosides in the wild-type (WT) and ΔypfI strains. The top panel is the UV trace at 254 nm of the WT strain. The middle (WT) and bottom (ΔypfI) panels are mass chromatograms at m/z 286 for detecting a proton adduct (MH⁺) of ac⁴C. Absence of ac⁴C in ΔypfI strain is indicated by the arrow. Asterisks indicate isotopic ions of guanosine. (B) Growth property of the ΔypfI/ΔdusC strain. Each deletion strain was serially diluted (1:10 dilutions) and then spotted onto LB plates and incubated at 37 and 24 °C for 2 days.

Figure 3

Sequence alignment of TmcA from γ-proteobacteria. Amino-acid sequence alignment of YpfI in E. coli (ECO) and homologues in other bacteria (STY, S. typhimurium LT2; YPE, Y. pestis; HIN, H. influenzae R3021; PMU, P. multocida; and VCH, V. cholerae) were aligned. Two domains (DUF699 and Acetyltransf_1) predicted by Pfam are indicated by black solid and dotted lines, respectively. The Walker A motif is indicated by grey line. The black and grey boxes indicate a degree of sequence similarity.

Figure 4

Reconstitution of ac⁴C formation in vitro using recombinant TmcA. (A) Mass spectrometric detection of ac⁴C formation in the unmodified tRNA^Met that was transcribed in vitro. After the reaction, the tRNA^Met was digested into nucleosides and analysed by LC/MS. Mass chromatograms at m/z 286 detected ac⁴C, which was reconstituted in vitro in the absence of both ATP and acetyl-CoA (second panel), in the presence of ATP (third panel), in the presence of acetyl-CoA (fourth panel) or in the presence of both ATP and acetyl-CoA (bottom panel). The ac⁴C appeared only when both substrates were present. (B) Mass spectrum of ac⁴C that was reconstituted in vitro. (C) In vitro reconstitution of ac⁴C formation in the presence (filled circle) or absence (open circle) of ATP, which was detected by liquid scintillation counting of ¹⁴C-acetyl group. (D) In vitro reconstitution of ac⁴C formation under various conditions, the radioactivity of which was visualized by an imaging analyser (FLA-7000, Fujifilm). The upper and lower panels show visualized radioactivity and ethidium bromide staining. The reaction was performed in the presence of ATP (lane 1), GTP (lane 7) and ADP (lane 6), in the absence of ATP (lane 3) and TmcA (lane 2). The mutant tRNA^Met, EM(C34G), whose C34 was replaced by G34 (lane 4) and tRNA^Ile2 (lane 5), were employed for ac⁴C reconstitution instead of tRNA^Met. (E) Gel-retardation analysis of tRNA^Met with recombinant TmcA. The polyacrylamide gel was stained by ethidium bromide (upper panel) and Coomassie brilliant blue (lower panel). Lane 1, TmcA (80 pmol); lane 2, tRNA^Met; lane 3, TmcA (40 pmol) and tRNA^Met; lane 4, TmcA (80 pmol) and tRNA^Met; lane 5, TmcA (160 pmol) and tRNA^Met; lane 6, TmcA (80 pmol) and tRNA^Ile2; lane 7, tRNA^Ile2. Amount of tRNA was 80 pmol in all conditions.

Figure 5

Mutation study of tRNA^Met and tRNA^Ile2 to investigate the positive and negative determinants of ac⁴C formation. (A) tRNA variants based on E. coli elongator tRNA^Met (left-hand side) and E. coli tRNA^Ile2 (right-hand side) that were used in this study. The numbering system of the tRNA is based on the tRNA compilation by Sprinzl and Vassilenko (2005). Arrows and boxes indicate the substitutions and deletions made in this study. EM and EI stand for E. coli elongator tRNA^Met and E. coli tRNA^Ile2, respectively. (B) Detection of ac⁴C formation in tRNA variants visualized by the imaging analyser. (C) Relative acetylation activities of a series of tRNA variants. Radioactivity of ¹⁴C-acetylation on each tRNA was quantified by FLA-7000 system (Fujifilm). Radioactivity of ac⁴C in EM(WT) was standardized as 100%. A full-colour version of this figure is available at The Embo Journal Online.

Figure 6

Recognition and discrimination, by TmcA and TilS, of two tRNAs with CAU anticodons. Schematic depiction of tRNA recognition by TmcA in comparison with TilS. In this picture, the anticodon regions of both tRNAs are not highlighted as positive elements for both enzymes, as this region is commonly recognized by both enzymes. TmcA strongly recognizes C27-G43 in the anticodon stem of tRNA^Met. In addition, C30-G40 and G44 in tRNA^Met have an important function for TmcA recognition. G27·U43 in tRNA^Ile2 exerts an effect as a negative determinant for TmcA recognition. On the other hand, TilS strongly recognizes two consecutive base pairs, C4-G69 and C5-G68, in the acceptor stem of tRNA^Ile2 for lysidine formation, whereas U4-A69 and A5-U68 in tRNA^Met exert an effect as negative determinants for TilS recognition.