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Acetate-dependent tRNA acetylation required for decoding fidelity in protein synthesis

Abstract

Modification of tRNA anticodons plays a critical role in ensuring accurate translation. N4-acetylcytidine (ac4C) is present at the anticodon first position (position 34) of bacterial elongator tRNAMet. Herein, we identified Bacillus subtilis ylbM (renamed tmcAL) as a novel gene responsible for ac4C34 formation. Unlike general acetyltransferases that use acetyl-CoA, TmcAL activates an acetate ion to form acetyladenylate and then catalyzes ac4C34 formation through a mechanism similar to tRNA aminoacylation. The crystal structure of TmcAL with an ATP analog reveals the molecular basis of ac4C34 formation. The ΔtmcAL strain displayed a cold-sensitive phenotype and a strong genetic interaction with tilS that encodes the enzyme responsible for synthesizing lysidine (L) at position 34 of tRNAIle to facilitate AUA decoding. Mistranslation of the AUA codon as Met in the ΔtmcAL strain upon tilS repression suggests that ac4C34 modification of tRNAMet and L34 modification of tRNAIle act cooperatively to prevent misdecoding of the AUA codon.

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Fig. 1: Identification of ac4C in B. subtilis tRNAeMet and ylbM responsible for ac4C formation.
Fig. 2: Mechanistic characterization of ac4C formation catalyzed by TmcAL.
Fig. 3: Crystal structure of B. subtilis TmcAL and mutational analysis.
Fig. 4: Mutational analysis of tRNAs to investigate the determinants for ac4C formation mediated by TmcAL.
Fig. 5: Genetic interaction between tmcAL and tilS and mistranslation at the AUA codon.
Fig. 6: ac4C34 and L34 modifications cooperatively prevent mistranslation of the AUA codon.

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Acknowledgements

We thank members of the Suzuki laboratory, especially S. Kimura, for fruitful discussion and many helpful suggestions. We also thank the beamline staff at BL-17A of the Photon Factory for technical assistance during data collection, and M. Miyata in Osaka City University for kindly giving us the culture of M. mobile. This work was supported by the Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (26113003, 26220205, 18H05272 to T.S.; 18K05430 to K.M.; and 26113002, 18H03980 to K.T.), by a JSPS Fellowship for Japanese Junior Scientists (to T.T.), and by the Noda Institute for Scientific Research (to A.S.).

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T.T. mainly performed the series of experiments. K.M. assisted biochemical and informatics works. Y.S. performed MS analysis. S.Y. and K.T. performed structural studies. A.S. assisted genetic works. All authors discussed the results. T.T. and T.S. wrote this paper. T.S. designed and supervised all the work.

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Correspondence to Tsutomu Suzuki.

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Taniguchi, T., Miyauchi, K., Sakaguchi, Y. et al. Acetate-dependent tRNA acetylation required for decoding fidelity in protein synthesis. Nat Chem Biol 14, 1010–1020 (2018). https://doi.org/10.1038/s41589-018-0119-z

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