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Supplementary Information Materials and Methods Supplementary Information Materials and methods Preparation of Proteins Plasmids for expression of full-length E. coli or H. sapiens AlaRS were constructed through PCR amplification of the desired region of the AlaRS genes with oligonucleotides containing NdeI-XhoI or NdeI-BamHI sites and ligated into pET20b to generate pET20b-EcAlaRS and pET20b-HsAlaRS. Plasmids for expression of mutant E. coli AlaRS (N303A, N303D, D400A, D400N, N303A/D400A) and H. sapiens AlaRS (N317A, N317D, D416A, D416N, N317A/D416A) were constructed by the standard Quikchange mutagenesis of pET20b-EcAlaRS or pET20b-HsAlaRS. These proteins were expressed in E. coli BL21 (DE3) cells (Stratagene) and purified by Ni-NTA affinity column (Qiagen) and a Q high performance column (GE Healthcare). All proteins were dialyzed against 5 mM Tris-HCl buffer, pH 8.0, 50 mM NaCl and 2 mM DTT. Preparation of tRNAs Transfer RNAs were produced by in vitro transcription. 10 mL transcription reactions were carried out at 37 °C for 2 hours in buffer (40 mM Tris-HCl, pH 8.0, 25 mM NaCl, 20 mM MgCl2, 2 mM 1,8- diaminooctane, 10 mM DTT) with T7 RNA polymerase (40 U/μL), and BstNI-linearized pUC18-tRNAAla plasmid DNA template (1 μM). The transcribed tRNAs were purified by a 8 M urea-denaturing PAGE gel or in combination with a DEAE column, annealed and concentrated. The quantity of tRNA was determined by A260. www.pnas.org/cgi/doi/10.1073/pnas.1807109115 Active Site Titration Assays Active site titration was performed at 25 °C in assay buffer (50 mM HEPES, pH 7.5, 20 mM KCl, 5 mM 32 MgCl2, 2 mM DTT, 0.05 U/mL dialyzed yeast inorganic pyrophosphatase) with γ- [P]-ATP (20 μM) and L- Ala (1 mM). Aliquots were quenched at different time points and spun in 96-well Multiscreen filter plates (Millipore) with activated charcoals as described previously29. The flow-through samples were counted in a MicroBeta plate reader (PerkinElmer). The amount of the released 32[P]-phosphate was used to determine the concentration of active enzymes. Aminoacylation Assays Aminoacylation with wild-type tRNAAla was performed by incubating purified enzyme (5 nM) in assay buffer (50 mM HEPES, pH 7.5, 20 mM KCl, 5 mM MgCl2, 2 mM DTT, 0.05 U/mL dialyzed yeast inorganic pyrophosphatase), with 4 mM ATP, 20 μM [3H]-L-Ala and 2 μM tRNAAla at 25 °C. For reactions with G3:C70 tRNAAla, 500 nM enzyme and 10 μM G3:C70 tRNAAla was used, at 37 °C. Aliquots were quenched and precipitated in 96-well Multiscreen filter plates as described previously29. After washing and elution by NaOH, samples were counted in a MicroBeta plate reader (PerkinElmer). Structural Modelling E. coli and H. sapiens AlaRS structures were first modeled using Modeller 8v2 (http://salilab.org/modeller/) based on the available E. coli AlaRS catalytic domain crystal structure and the newly solved A. fulgidus AlaRS•tRNAAla complex structure and on multiple sequence alignments. The initial docking of tRNAAla to E. coli and H. sapiens AlaRS structures was determined according to the structural superimposition of the N-terminal catalytic fragments. The conformation of the E. coli AlaRS Mid2 subdomain and the interface residues in both E. coli and H. sapiens structures were slightly adjusted for the tRNA binding. Supplementary Figure 1 Figure S1. Comparison of E. coli, H. sapiens and A. fulgidus catalytic fragment structures. a, Superposition of the E. coli and A. fulgidus catalytic fragment structures. b, Superposition of the H. sapiens and A. fulgidus catalytic fragment structures. The A. fulgidus catalytic fragment is colored in teal, light blue, and dark blue for aminoacylation, Mid1, and Mid2 domains, respectively. E. coli and H. sapiens catalytic fragments are colored gray with the Mid2 subdomains highlighted in red. The A. fulgidus Mid1 domain contains a large archaea-specific insertion. Supplementary Figure 2 Figure S2. Sequence alignments of full-length AlaRS from A. fulgidus, E. coli and H. sapiens. The common structure consisting of an Asn and an Asp residue for the recognition of G3:U70 tRNAAla by AlaRS is highlighted. The numbering is according to the E. coli AlaRS sequence. Supplementary Figure 3 a b c d Figure S3. Aminoacylation activities of wild-type (WT) and mutant AlaRS enzymes towards tRNAAla and G3:C70 tRNAAla. a-b, Aminoacylation of G3:U70 tRNAAla (a) and G3:C70 tRNAAla (b) by E. coli AlaRS enzymes. c-d, Aminoacylation of G3:U70 tRNAAla (c) and G3:C70 tRNAAla (d) by human AlaRS enzymes. Aminoacylation of G3:U70 tRNAAla was carried out with 5 nM of each enzyme at 25 °C, while that of G3:C70 tRNAAla was carried out with 500 nM of each enzyme at 37 °C. Assays were performed at pH 7.5. Error bars represent the s.e.m. of triplicate experiments. Supplementary Table 1. Verified AlaRS sequences with conserved Asn + Asp structure for G:U recognition. Accession Entry name Kingdom Gene names Organism Length 1 A0B6X3 SYA_METTP Archaea alaS Mthe_0657 Methanosaeta thermophila (strain DSM 6194 / PT) (Methanothrix thermophila913 (strain DSM 6194 / PT)) 2 A0RX10 SYA_CENSY Archaea alaS CENSYa_1254 Cenarchaeum symbiosum 894 3 A1RT13 SYA_PYRIL Archaea alaS Pisl_0919 Pyrobaculum islandicum (strain DSM 4184 / JCM 9189) 892 4 A1RWQ1 SYA_THEPD Archaea alaS Tpen_0221 Thermofilum pendens (strain Hrk 5) 907 5 A2BN65 SYA_HYPBU Archaea alaS Hbut_1607 Hyperthermus butylicus (strain DSM 5456 / JCM 9403) 921 6 A2ST90 SYA_METLZ Archaea alaS Mlab_1380 Methanocorpusculum labreanum (strain ATCC 43576 / DSM 4855 / Z) 913 7 A3CVP2 SYA_METMJ Archaea alaS Memar_1513 Methanoculleus marisnigri (strain ATCC 35101 / DSM 1498 / JR1) 915 8 A3DNI3 SYA_STAMF Archaea alaS Smar_1097 Staphylothermus marinus (strain ATCC 43588 / DSM 3639 / F1) 909 9 A3MXP1 SYA_PYRCJ Archaea alaS Pcal_1993 Pyrobaculum calidifontis (strain JCM 11548 / VA1) 896 10 A4FZA5 SYA_METM5 Archaea alaS MmarC5_1241 Methanococcus maripaludis (strain C5 / ATCC BAA-1333) 892 11 A4WN07 SYA_PYRAR Archaea alaS Pars_2228 Pyrobaculum arsenaticum (strain DSM 13514 / JCM 11321) 892 12 A5UKU6 SYA_METS3 Archaea alaS Msm_0619 Methanobrevibacter smithii (strain PS / ATCC 35061 / DSM 861) 897 13 A6US09 SYA_METVS Archaea alaS Mevan_1384 Methanococcus vannielii (strain SB / ATCC 35089 / DSM 1224) 892 14 A6UUN3 SYA_META3 Archaea alaS Maeo_0621 Methanococcus aeolicus (strain Nankai-3 / ATCC BAA-1280) 910 15 A6VJ32 SYA_METM7 Archaea alaS MmarC7_1395 Methanococcus maripaludis (strain C7 / ATCC BAA-1331) 892 16 A7I7S4 SYA_METB6 Archaea alaS Mboo_1267 Methanoregula boonei (strain 6A8) 914 17 A8A8X8 SYA_IGNH4 Archaea alaS Igni_0196 Ignicoccus hospitalis (strain KIN4/I / DSM 18386 / JCM 14125) 901 18 A8MBI2 SYA_CALMQ Archaea alaS Cmaq_1898 Caldivirga maquilingensis (strain DSMZ 13496 / IC-167) 899 19 A9A565 SYA_NITMS Archaea alaS Nmar_0368 Nitrosopumilus maritimus (strain SCM1) 889 20 A9A6Q2 SYA_METM6 Archaea alaS MmarC6_0513 Methanococcus maripaludis (strain C6 / ATCC BAA-1332) 892 21 B0R7I0 SYA_HALS3 Archaea alaS OE4198F Halobacterium salinarum (strain ATCC 29341 / DSM 671 / R1) 929 22 B1L762 SYA_KORCO Archaea alaS Kcr_1545 Korarchaeum cryptofilum (strain OPF8) 895 23 B1YDH4 SYA_THENV Archaea alaS Tneu_0900 Thermoproteus neutrophilus (strain DSM 2338 / JCM 9278 / V24Sta) 892 24 B5IBA6 B5IBA6_ACIB4 Archaea alaS2 ABOONEI_543 Aciduliprofundum boonei (strain DSM 19572 / T469) 882 25 B5ICY4 B5ICY4_ACIB4 Archaea alaS1 Aboo_1447 ABOONEI_1022Aciduliprofundum boonei (strain DSM 19572 / T469) 882 26 B8D5P5 B8D5P5_DESK1 Archaea alaS DKAM_1100 Desulfurococcus kamchatkensis (strain 1221n / DSM 18924) 908 27 B8GHL6 B8GHL6_METPE Archaea alaS Mpal_1287 Methanosphaerula palustris (strain ATCC BAA-1556 / DSM 19958 / E1-9c)913 28 B9AGG4 B9AGG4_METSM Archaea alaS METSMIALI_01468 Methanobrevibacter smithii DSM 2375 897 29 B9LUG4 SYA_HALLT Archaea alaS Hlac_0719 Halorubrum lacusprofundi (strain ATCC 49239 / DSM 5036 / JCM 8891 926/ ACAM 34) 30 C3MR87 SYA_SULIL Archaea alaS LS215_1905 Sulfolobus islandicus (strain L.S.2.15 / Lassen #1) 900 31 C3MXH6 SYA_SULIM Archaea alaS M1425_1765 Sulfolobus islandicus (strain M.14.25 / Kamchatka #1) 900 32 C3MZC2 SYA_SULIA Archaea alaS M1627_1883 Sulfolobus islandicus (strain M.16.27) 900 33 C3N7E3 SYA_SULIY Archaea alaS YG5714_1881 Sulfolobus islandicus (strain Y.G.57.14 / Yellowstone #1) 900 34 C3NG91 SYA_SULIN Archaea alaS YN1551_1044 Sulfolobus islandicus (strain Y.N.15.51 / Yellowstone #2) 900 35 C4KIK1 SYA_SULIK Archaea alaS M164_1812 Sulfolobus islandicus (strain M.16.4 / Kamchatka #3) 900 36 C7NT23 C7NT23_HALUD Archaea alaS Huta_1929 Halorhabdus utahensis (strain DSM 12940 / JCM 11049 / AX-2) 926 37 C7P221 C7P221_HALMD Archaea alaS Hmuk_1124 Halomicrobium mukohataei (strain ATCC 700874 / DSM 12286 / JCM 9738924 / NCIMB 13541) (Haloarcula mukohataei) 38 C7P7U1 C7P7U1_METFA Archaea alaS Mefer_0805 Methanocaldococcus fervens (strain DSM 4213 / JCM 157852 / AG86) (Methanococcus892 fervens) 39 C9RF77 C9RF77_METVM Archaea alaS Metvu_0364 Methanocaldococcus vulcanius (strain ATCC 700851 / DSM 12094 / M7)895 (Methanococcus vulcanius) 40 D0KS30 D0KS30_SULS9 Archaea alaS Ssol_1313 Sulfolobus solfataricus (strain 98/2) 900 41 D1JFB4 D1JFB4_9ARCH Archaea alaS BSM_06990 Uncultured archaeon 919 42 D2PDA7 D2PDA7_SULID Archaea alaS LD85_2022 Sulfolobus islandicus (strain L.D.8.5 / Lassen #2) 900 43 D2RFS9 D2RFS9_ARCPA Archaea alaS Arcpr_0078 Archaeoglobus profundus (strain DSM 5631 / JCM 9629 / NBRC 100127902 / Av18) 44 D2RSW0 D2RSW0_HALTV Archaea alaS Htur_0033 Haloterrigena turkmenica (strain ATCC 51198 / DSM 5511 / NCIMB 13204925 / VKM B-1734) (Halococcus turkmenicus) 45 D2ZN69 D2ZN69_METSM Archaea alaS METSMIF1_02273 Methanobrevibacter
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