Mistranslation arising from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences1,2,3. Throughout evolution, two editing checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly1,2. One checkpoint is the AlaRS editing centre, and the other is from widely distributed AlaXps—free-standing, genome-encoded editing proteins that clear Ser-tRNAAla. The paradox of misincorporating both a smaller (glycine) and a larger (serine) amino acid suggests a deep conflict for nature-designed AlaRS. Here we show the chemical basis for this conflict. Nine crystal structures, together with kinetic and mutational analysis, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the α-amino group of the bound amino acid by using an acidic residue. This design, dating back more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently because no better architecture for the recognition of alanine could be found, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Protein Data Bank
Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession codes 3hxu (WT/Ala-SA), 3hxv (WT/Gly-SA), 3hxw (WT/Ser-SA), 3hxx (WT/AMP-PCP/Mg(II)), 3hxy (WT/Ala-AMP/PCP/AMP-PCP/Mg(II)), 3hxz (G237A/Ala-SA), 3hy0 (G237A/Gly-SA) and 3hy1 (G237A-apo and G237A/Ser-SA).
Beebe, K., Ribas De Pouplana, L. & Schimmel, P. Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability. EMBO J. 22, 668–675 (2003)
Lee, J. W. et al. Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration. Nature 443, 50–55 (2006)
Beebe, K., Mock, M., Merriman, E. & Schimmel, P. Distinct domains of tRNA synthetase recognize the same base pair. Nature 451, 90–93 (2008)
Carter, C. W. Cognition, mechanism, and evolutionary relationships in aminoacyl-tRNA synthetases. Annu. Rev. Biochem. 62, 715–748 (1993)
Giege, R. The early history of tRNA recognition by aminoacyl-tRNA synthetases. J. Biosci. 31, 477–488 (2006)
Norris, A. T. & Berg, P. Mechanism of aminoacyl RNA synthesis: studies with isolated aminoacyl adenylate complexes of isoleucyl RNA synthetase. Proc. Natl Acad. Sci. USA 52, 330–337 (1964)
Eldred, E. W. & Schimmel, P. R. Rapid deacylation by isoleucyl transfer ribonucleic acid synthetase of isoleucine-specific transfer ribonucleic acid aminoacylated with valine. J. Biol. Chem. 247, 2961–2964 (1972)
Boniecki, M. T., Vu, M. T., Betha, A. K. & Martinis, S. A. CP1-dependent partitioning of pretransfer and posttransfer editing in leucyl-tRNA synthetase. Proc. Natl Acad. Sci. USA 105, 19223–19228 (2008)
Fersht, A. R. Enzyme Structure and Mechanism (Freeman, 1977)
Nureki, O. et al. Structural basis for amino acid and tRNA recognition by class I aminoacyl-tRNA synthetases. Cold Spring Harb. Symp. Quant. Biol. 66, 167–173 (2001)
Fersht, A. R. Sieves in sequence. Science 280, 541 (1998)
Fukai, S. et al. Structural basis for double-sieve discrimination of l-valine from l-isoleucine and l-threonine by the complex of tRNAVal and valyl-tRNA synthetase. Cell 103, 793–803 (2000)
Tsui, W. C. & Fersht, A. R. Probing the principles of amino acid selection using the alanyl-tRNA synthetase from Escherichia coli . Nucleic Acids Res. 9, 4627–4637 (1981)
Ahel, I., Korencic, D., Ibba, M. & Söll, D. Trans-editing of mischarged tRNAs. Proc. Natl Acad. Sci. USA 100, 15422–15427 (2003)
Chong, Y. E., Yang, X. L. & Schimmel, P. Natural homolog of tRNA synthetase editing domain rescues conditional lethality caused by mistranslation. J. Biol. Chem. 283, 30073–30078 (2008)
Ling, J. et al. Resampling and editing of mischarged tRNA prior to translation elongation. Mol. Cell 33, 654–660 (2009)
Guo, M. et al. The C-Ala domain brings together editing and aminoacylation functions on one tRNA. Science 325, 744–747 (2009)
Sokabe, M., Okada, A., Yao, M., Nakashima, T. & Tanaka, I. Molecular basis of alanine discrimination in editing site. Proc. Natl Acad. Sci. USA 102, 11669–11674 (2005)
Ho, B. K. & Gruswitz, F. HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, 49 (2008)
Arnez, J. G. & Moras, D. Structural and functional considerations of the aminoacylation reaction. Trends Biochem. Sci. 22, 211–216 (1997)
Davis, M. W., Buechter, D. D. & Schimmel, P. Functional dissection of a predicted class-defining motif in a class II tRNA synthetase of unknown structure. Biochemistry 33, 9904–9911 (1994)
Jakubowski, H. in The Aminoacyl-tRNA Synthetases (eds Ibba, M., Francklyn, C. & Cusack, S.) 384–396 (Eurekah, 2005)
Shi, J. P., Musier-Forsyth, K. & Schimmel, P. Region of a conserved sequence motif in a class II tRNA synthetase needed for transfer of an activated amino acid to an RNA substrate. Biochemistry 33, 5312–5318 (1994)
Xin, Y., Li, W. & First, E. A. Stabilization of the transition state for the transfer of tyrosine to tRNATyr by tyrosyl-tRNA synthetase. J. Mol. Biol. 303, 299–310 (2000)
Sankaranarayanan, R. et al. Zinc ion mediated amino acid discrimination by threonyl-tRNA synthetase. Nature Struct. Biol. 7, 461–465 (2000)
First, E. A. in The Aminoacyl-tRNA Synthetases (eds Ibba, M., Francklyn, C. & Cusack, S.) 328–352 (Eurekah, 2005)
Belrhali, H. et al. Crystal structures at 2.5 angstrom resolution of seryl-tRNA synthetase complexed with two analogs of seryl adenylate. Science 263, 1432–1436 (1994)
Cieslik, M. & Derewenda, Z. S. The role of entropy and polarity in intermolecular contacts in protein crystals. Acta Crystallogr. D 65, 500–509 (2009)
Itoh, Y. et al. Crystallographic and mutational studies of seryl-tRNA synthetase from the archaeon Pyrococcus horikoshii . RNA Biol. 5, 169–177 (2008)
Swairjo, M. A. & Schimmel, P. R. Breaking sieve for steric exclusion of a noncognate amino acid from active site of a tRNA synthetase. Proc. Natl Acad. Sci. USA 102, 988–993 (2005)
Malde, A. K. & Mark, A. E. Binding and enantiomeric selectivity of threonyl-tRNA synthetase. J. Am. Chem. Soc. 131, 3848–3849 (2009)
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)
CCP4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
We thank R. J. Read for pointing out the errors in the previous AlaRS–ligand structures and for crystallographic discussions; and G. J. Kleywegt, Z. Otwinowski and A. Perrakis for technical assistance. X-ray diffraction data were collected at Stanford Synchrotron Radiation Laboratory (SSRL) beamlines 7-1, 9-1 and 11-1. This work was supported by grant GM 15539 from the National Institutes of Health and by a fellowship from the National Foundation for Cancer Research.
Author Contributions M.G., X.-L.Y. and P.S. designed the experiments. M.G., Y.E.C., R.S. and K.B. performed the experiments. M.G. and Y.E.C. analysed the data. M.G., Y.E.C., X.-L.Y. and P.S. wrote the paper. All authors discussed the results and commented on the manuscript.
This file contains Supplementary Figures 1-8 with Legends. (PDF 5485 kb)
This file contains a summary of the data collection and refinement statistics of the nine structures. (PDF 192 kb)
This movie file shows that serine paradox is caused by AlaRS recognition dilemma (see Supplementary Figures file for full Legend). (MOV 6540 kb)
This movie file shows the alanyl-adenylate formation mechanism (see Supplementary Figures file for full Legend). (MOV 4436 kb)
About this article
Cite this article
Guo, M., Chong, Y., Shapiro, R. et al. Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma. Nature 462, 808–812 (2009). https://doi.org/10.1038/nature08612
How evolution shapes enzyme selectivity – lessons from aminoacyl‐tRNA synthetases and other amino acid utilizing enzymes
The FEBS Journal (2020)
The mechanism of β-N-methylamino-l-alanine inhibition of tRNA aminoacylation and its impact on misincorporation
Journal of Biological Chemistry (2020)
LOTTE-seq (Long hairpin oligonucleotide based tRNA high-throughput sequencing): specific selection of tRNAs with 3’-CCA end for high-throughput sequencing
RNA Biology (2020)
PLOS Genetics (2019)
Structure-Guided Enhancement of Selectivity of Chemical Probe Inhibitors Targeting Bacterial Seryl-tRNA Synthetase
Journal of Medicinal Chemistry (2019)