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Aminoacylation of tRNA with phosphoserine for synthesis of cysteinyl-tRNACys

Abstract

Cysteinyl-tRNACys (Cys-tRNACys) is required for translation and is typically synthesized by cysteinyl-tRNA synthetase (CysRS). However, Methanocaldococcus jannaschii synthesizes Cys-tRNACys by an indirect pathway, whereby O-phosphoseryl–tRNA synthetase (SepRS) acylates tRNACys with phosphoserine (Sep), and Sep-tRNA–Cys-tRNA synthase (SepCysS) converts the tRNA-bound phosphoserine to cysteine. We show here that M. jannaschii SepRS differs from CysRS by recruiting the m1G37 modification as a determinant for aminoacylation, and in showing limited discrimination against mutations of conserved nucleotides. Kinetic and binding measurements show that both SepRS and SepCysS bind the reaction intermediate Sep-tRNACys tightly, and these two enzymes form a stable binary complex that promotes conversion of the intermediate to the product and sequesters the intermediate from binding to elongation factor EF-1α or infiltrating into the ribosome. These results highlight the importance of the protein binary complex for efficient synthesis of Cys-tRNACys.

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Figure 1: Aminoacylation of tRNACys transcript by M. jannaschii SepRS.
Figure 2: Burst kinetics of aminoacylation.
Figure 3: Binding of Sep-tRNACys to SepCysS and EF-1α-GTP.
Figure 4: The SepRS–SepCysS binary complex.
Figure 5: Binding of Sep-tRNACys to the SepRS–SepCysS complex.

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Acknowledgements

Supported by the US National Institutes of Health grant GM066267 to Y.-M.H. We thank S. Kim, T. Christian and H. Gamper for assistance with experiments, and J. Perona and H. Gamper for comments on the manuscript.

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Authors

Contributions

C.-M.Z., C.L., S.S. and Y.-M.H. designed experiments; C.-M.Z. and C.L. performed experiments; and Y.-M.H. wrote the manuscript.

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Correspondence to Ya-Ming Hou.

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Supplementary Figures 1–3 and Supplementary Table 1 (PDF 1102 kb)

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Zhang, CM., Liu, C., Slater, S. et al. Aminoacylation of tRNA with phosphoserine for synthesis of cysteinyl-tRNACys. Nat Struct Mol Biol 15, 507–514 (2008). https://doi.org/10.1038/nsmb.1423

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