SUPPLEMENTARY INFORMATION. An orthogonal ribosome-trnas pair by the engineering of

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1 SUPPLEMENTARY INFORMATION An orthogonal ribosome-trnas pair by the engineering of peptidyl transferase center Naohiro Terasaka 1 *, Gosuke Hayashi 1 *, Takayuki Katoh 1, and Hiroaki Suga 1,2 1 Department of Chemistry, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo , Japan 2 JST, CREST, The University of Tokyo, Tokyo , Japan *These authors equally contributed to this work Present address: Research Center for Advanced Science and Technology, The University of Tokyo, Komaba, Meguro-ku, Tokyo , Japan 1

2 Supplementary Results Supplementary Figure 1 Amino acid substrates used in this study. The abbreviations of Fph, Aly, and Anv represent N-(5-FAM)-L-phenylalanine, L-acetyllysine, and L-azidonorvaline, respectively. Carboxyl groups were activated using cyanomethyl ester (CME) or 3,5-dinitrobenzyl ester (DBE). 2

3 Supplementary Figure 2 Aminoacylation of trnas-nna by flexizyme-nnu. NN in flexizymes (dfx and efx) forms two consecutive Watson Crick base pairs with NN in trnas. Amino acids (AA) bearing non-aromatic or aromatic sidechains are esterified with a designated leaving group (LG), 3,5-dinitrobenzyl ester (DBE) for dfx or cyanomethyl ester (CME) for efx. 3

4 Supplementary Figure 3 Secondary structures of transcribed (t.s.) trnas compared with native trnas of E. coli. The figure presents the secondary structures of trna fmet CAU (a), trna AsnE2 NNN (b), trna GluE2 NNN (c), trna Lys CUU (d), trna Asp GUC (e), and trna Tyr GUA (f). 4

5 5

6 Supplementary Figure 4 A model study of aminoacylation of trnas-nna derived from trna AsnE2. trnas were acylated by dfx in the presence of Lys-DBE (a), and efx in the presence of Tyr-CME (b). Acid-PAGE separated the bands of aa-trna (upper) and uncharged trna (lower). The data were generated from a sample of the end product of aminoacylation reaction. Note that the mobility of flexizymes was different depending on the 3'-terminal mutations. 6

Supplementary Figure 5 Efficiency of flexizyme-catalyzed aminoacylation with proteinogenic (Tyr, Lys, and Asp) and non-proteinogenic (Fph, Aly, and Anv) amino acids.

7 Supplementary Figure 5 Efficiency of flexizyme-catalyzed aminoacylation with proteinogenic (Tyr, Lys, and Asp) and non-proteinogenic (Fph, Aly, and Anv) amino acids. The trnas-cca, -GCA, -CGA, -GGA and -CUA were acylated by the compensatory flexizymes, dfx/efx-ggu, -GCU, -CGU, -CCU and -AGU. The data represent the average of three independent reactions. The error bars represent the standard deviation. The data were generated from a sample of the end product of aminoacylation reaction. 7

8 Supplementary Figure 6 MALDI-TOF MS analysis of heptapeptide-1 synthesized using cognate and non-cognate ribosome trna pairs. Lane numbers are those of the Tricine-SDS-PAGE gel described in Figure 2a. C and O denote calculated and observed mass values, respectively. indicates a potassium adduct of heptapeptide-1. indicates the molecular mass value consistent with FphLysTyrLysLysTyr, which was presumably produced by peptidyl-trna drop-off 26. The identity of major peak in lane 16 is unknown; however, since this peaks as well as other minor peaks were also present in the sample of G2251C/G225C/G2553C-ribosome alone, it could be originated from a contaminant of the G2251C/G225C/G2553C-ribosome preparation. The data were generated from a sample of the end product of translation reaction. 8

9 Supplementary Figure 7 Translational activity of pairs of WT ribosome trna using trnas AsnE2 as elongator trnas. (a) Time-course analysis of heptapeptide-1 production. The data represent the average of three independent reactions and error bars represent the standard deviation of the individual measurements. (b) MALDI-TOF MS analysis of heptapeptide-1. C and O denote calculated and observed mass values, respectively. indicates a potassium adduct of heptapeptide-1. Other minor peaks were also present in the non-templated translation product likely originating from the translation system. The data were generated from a sample of the end product of translation reaction. 9

Supplementary Figure 8 Gel-shift assay for ternary complex of elongation factor Tu (EF-Tu), guanosine

Tyrosine was charged onto trna AsnE2 GUA and trna GluE2 GUA, and lysine was charged onto trna AsnE2 CUU

Band intensities of the ternary complexes were normalized relative to EF-Tu, GTP and aa-trnas-cca

10 Supplementary Figure 8 Gel-shift assay for ternary complex of elongation factor Tu (EF-Tu), guanosine triphosphate (GTP) and aa-trnas-cca, GCA, CGA and GGA. Tyrosine was charged onto trna AsnE2 GUA and trna GluE2 GUA, and lysine was charged onto trna AsnE2 CUU and trna GluE2 CUU. Band intensities of the ternary complexes were normalized relative to EF-Tu, GTP and aa-trnas-cca complexes. The affinities of aa-trnas-cca, GCA, CGA and GGA to EF-Tu were comparable, which was coincident with the previous report The values of the relative intensity of the ternary complex (y-axis) were derived from a single experimental set. 10

11 Supplementary Figure 9 Illustration of compatibility and orthogonality of the ribosome trnas mutant pairs. Line thickness indicates the compatibility of translational activity between each ribosome trna pair. 11

12 Supplementary Figure 10 Primer extension analysis of rrnas extracted from MS2-tagged ribosomes purified by FPLC. (a) A primer that is complementary to the bases of in 23S rrna was used for the detection of mutations of 2251 and (b) A primer that is complementary to the bases of in 23S rrna was used for the detection of mutation of MS2-taggd ribosomes bearing mutations produced different ddgtp or ddttp stops from those produced by WT ribosome. The abundance of tagged ribosomes relative to untagged wildtype in each population was shown below the gel. The calculated values of abundance were derived from a single experimental data set. 12

13 13

14 Supplementary Figure 11 Translational activity of pairs of ribosome trna mutants bearing CCA, GCA, CGA, GGA and CUA-3 ends. trnas fmet were used as initiator trnas and trnas GluE2 as elongator trnas. (a) The entire image of the tricine-sds-page analysis of a heptapeptide-1 (FphLysTyrLysLysTyrLys) synthesized using pairs of ribosome trna mutants. This image showed the result of the end of the translation reaction. Two bands were observed for Fph (indicated by Fph1 and Fph2), presumably due to the difference in its protonation forms. The bands above and below of Fph1 showed truncated peptides generated by peptidyl-trna drop-off 26, which were also observed for the wildtype control of lane 1. (b) Time-course analysis of heptapeptide-1 production by active pairs of ribosomes and trnas. Lane numbers are those of the Tricine-SDS-PAGE gel described in Supplementary Figure 11a, in which the solid line in cyan indicates the reaction of a cognate pairs of the wildtype ribosome trnas-cca (the identical result shown in Figure 2b) while the dashed line in yellow shows that of a non-cognate pair G2253C-ribosome trnas-cca. The data represent the mean value of each sample (n = 3) and error bars show the standard deviations obtained from independent measurements. (c) MALDI-TOF MS analysis of heptapeptide-1 synthesized using a non-cognate pair G2253C-ribosome trnas-cca. Lane numbers are those of the Tricine-SDS-PAGE gel described in Supplementary Figure 11a. C and O denote calculated and observed mass values, respectively. indicates the molecular mass value consistent with FphLysTyrLysLysTyr, which could be one of the bands corresponding to the truncated peptides appeared in the tricine-sds-page. The data shown in Supplementary Figure 11a and c were generated from a sample of the end product of translation reaction. 14

15 Supplementary Figure 12 Two genetic codes programmed for simultaneous expression of two distinct peptides from a single mrna sequence. (a) WT-code. This code comprises the wildtype ribosome trnas-cca pair. (b) OR-code. This code comprises the G2251C/G2553C-ribosome trnas-cga pair. 15

16 Supplementary Figure 13 Time-course analysis about simultaneous expression shown in Figure 3c. Production of heptapeptide-2 in lane 1 is shown as black dashed line, lane 2 purple solid line, and lane 5 cyan solid line. Production of heptapeptide-3 in lane 3 is shown as brown dashed line, lane 4 yellow solid line, and lane 5 red solid line. Lane numbers are those described in Figure 3b. The data represent the average of three independent reactions, and the error bars represent the standard deviation of the individual measurements. 16

17 17

18 Supplementary Figure 14 Time-course analysis of heptapeptide production with an elevated concentration of EF-Tu. The concentration of EF-Tu was increased to 20 M from 10 M which was the condition in the experiments in Fig. 3. (a) Production of heptapeptide-2 when wildype ribosome coexisted with cognate aa-trnas-cca and noncognate aa-trna-cga with 20 M EF-Tu/Ts were shown as green dashed line. (b) Production of heptapeptide-3 when orthogonal ribosome coexisted with its noncognate and cognate aa-trnas with 20 M EF-Tu/Ts were shown as pink dashed line. (c) Production of heptapeptide-2 and -3 when both ribosomes with all aa-trnas coexisted with 20 M EF-Tu/Ts were shown as blue and orange dashed lines. The other lines represent the same result as those shown in Figure 3c. Lane numbers are those described in Figure 3b. The data represent the average of three independent reactions, and the error bars represent the standard deviation of the individual measurements. 18

Supplementary Figure 15 Simultaneous expression of two heptapeptides from a single mrna template according to the two artificially programmed genetic codes.

The pair of wildtype ribosome trnas-cca and G2251C/G2553C-ribosome trna-cga generated FphLysAspLysLysAspLys (heptapeptide-4) and FphLysAlyLysLysAlyLys (heptapeptide-5), respectively, according to

19 Supplementary Figure 15 Simultaneous expression of two heptapeptides from a single mrna template according to the two artificially programmed genetic codes. (a) Peptide translation from a single mrna sequence in a single reaction mixture. The pair of wildtype ribosome trnas-cca and G2251C/G2553C-ribosome trna-cga generated FphLysAspLysLysAspLys (heptapeptide-4) and FphLysAlyLysLysAlyLys (heptapeptide-5), respectively, according to their respective genetic codes. (b) Tricine-SDS-PAGE analysis of the respective heptapeptides, 4 and 5. The intense bands appeared below the band of heptapeptide-5 are Fph as discussed in Supplementary Figure 11a. Other faint based are truncated peptides generated by peptidyl-trna drop-off 26. (c) MALDI-TOF MS of the peptides isolated by gel filtration. Calculated (C) and observed mass (O) values are shown in the right panel of the spectra. indicates the potassium adduct of heptapeptide-4, indicates the potassium adduct of heptapeptide-5, *species also present in the no template translation product and were likely derived from the translation mixture. The data shown in Supplementary Figure 15b and c were generated from a sample of the end product of translation reaction. 19

20 Supplementary Figure 16 The entire image of gel shown in Figure 2a. The band assignments are the same as Supplementary Figure 11a. 20

Supplementary Figure 17 The entire image

The intense bands appeared below the band

21 Supplementary Figure 17 The entire image of gel shown in Figure 3b. Tricine-SDS-PAGE analysis of the respective heptapeptides-4 and -5. The intense bands appeared below the band of heptapeptide-5 are Fph as discussed in Supplementary Figure 11a. Other faint based are truncated peptides generated by peptidyl-trna drop-off

22 Supplementary Table 1 Primers Sequence(5' to 3') GGCGTAATACGACTCACTATAG GTAATACGACTCACTATAGGATCGAAAGATTTCCGC AACGCCATGTACCCTTTCGGGGATGCGGAAATCTTTCGATCC AACGCTAATCCCCTTTCGGGGCCGCGGAAATCTTTCGATCC AC(M)CTAACGCCATGTACCCTTTCGGG AC(M)CTAACGCTAATCCCCTTTCGGG AG(M)CTAACGCCATGTACCCTTTCGGG AG(M)CTAACGCTAATCCCCTTTCGGG AC(M)GTAACGCCATGTACCCTTTCGGG AC(M)GTAACGCTAATCCCCTTTCGGG AG(M)GTAACGCCATGTACCCTTTCGGG AG(M)GTAACGCTAATCCCCTTTCGGG AC(M)TTAACGCCATGTACCCTTTCGGG AC(M)TTAACGCTAATCCCCTTTCGGG GTAATACGACTCACTATACGCGGGGTGGAGCAGCCTGGTAGCTCGTCGG GAACCGACGATCTTCGGGTTATGAGCCCGACGAGCTACCAGGCT TTGCGGGGGCCGGATTTGAACCGACGATCTTCGGG GGCGTAATACGACTCACTATAC TG(M)GTTGCGGGGGCCGGATTTG TG(M)CTTGCGGGGGCCGGATTTG TC(M)GTTGCGGGGGCCGGATTTG TC(M)CTTGCGGGGGCCGGATTTG TA(M)GTTGCGGGGGCCGGATTTG GGCGTAATACGACTCACTATAGTCCCCTTCGTCTAGA CGTCCCCTAGGGGATTCGAACCCCTGTTACCGCC TATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTCT GAACCCCTGTTACCGCCTTAAGAGGGCGGTGTCCTGG TATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCCTGT GAACCCCTGTTACCGCCTTTACAGGGCGGTGTCCTGG TATAGTCCCCTTCGTCTAGAGGCCCAGGACACCGCCTTGT GAACCCCTGTTACCGCCTTGACAAGGCGGTGTCCTGG TG(M)GCGTCCCCTAGGGGATTC used for dfx, efx, trna, DNA template dfx-nnu and efx-nnu dfx-nnu efx-nnu dfx-ggu efx-ggu dfx-gcu efx-gcu dfx-cgu efx-cgu dfx-ggu efx-ggu dfx-agu efx-agu trna fmet CAU-NNA trna fmet CAU-NNA trna fmet CAU-NNA trna fmet CAU-NNA trna fmet CAU-CCA trna fmet CAU -GCA trna fmet CAU -CGA trna fmet CAU -GGA trna fmet CAU -CUA trna GluE2 NNN-NNA trna GluE2 NNN-NNA trna GluE2 CUU-NNA trna GluE2 CUU-NNA trna GluE2 GUA-NNA trna GluE2 GUA-NNA trna GluE2 GUC-NNA trna GluE2 GUC-NNA trna GluE2 NNN-CCA 22

23 Sequence(5' to 3') TG(M)CCGTCCCCTAGGGGATTC TC(M)GCGTCCCCTAGGGGATTC TC(M)CCGTCCCCTAGGGGATTC TA(M)GCGTCCCCTAGGGGATTC TATAGGGTCGTTAGCTCAGTTGGTAGAGCAGTTGACTCTTAATC GGCGTAATACGACTCACTATAGGGTCGTTAGCTCAGTTG TTCGAACCTGCGACCAATTGATTAAGAGTCAACTGCTCTACC TGGGTCGTGCAGGATTCGAACCTGCGACCAATTGATT TG(M)GTGGGTCGTGCAGGATTCG TC(M)GTGGGTCGTGCAGGATTCG TATAGGAGCGGTAGTTCAGTCGGTTAGAATACCTGCCTGT GGCGTAATACGACTCACTATAGGAGCGGTAGTTCAGTC GAACCCGCGACCCCCTGCGTGACAGGCAGGTATTCTAACCG CGGAACGGACGGGACTCGAACCCGCGACCCCC TG(M)GCGGAACGGACGGGACT AGGTGGGGTTCCCGAGCGGCCAAAGGGAGCAGACTGTAAATCT GGCGTAATACGACTCACTATAGGTGGGGTTCCCGAG GAACCTTCGAAGTCTGTGACGGCAGATTTACAGTCTGCTCCCT TGGTGGGGGAAGGATTCGAACCTTCGAAGTCTGTGA TG(M)GTGGTGGGGGAAGGATTCG GTAATACGACTCACTATAGGCTCTGTAGTTCAGTCGGTAGAACGGCGGA CGGCTCTGACTGGACTCGAACCAGTGACATACGGA GAACCAGTGACATACGGATTAAGAGTCCGCCGTTCTACCGACT GAACCAGTGACATACGGATTTACAGTCCGCCGTTCTACCGACT GAACCAGTGACATACGGATTGACAGTCCGCCGTTCTACCGACT TG(M)GCGGCTCTGACTGGACTC TG(M)CCGGCTCTGACTGGACTC TC(M)GCGGCTCTGACTGGACTC TC(M)CCGGCTCTGACTGGACTC TA(M)GCGGCTCTGACTGGACTC ATACGACTCACTATAGGGCTTTAATAAGGAGAAAAACATG GGCGTAATACGACTCACTATAGGGCTTT GTACTTCTTGTACTTCATGTTTTTCTCCTTATTAAAGCC used for trna GluE2 NNN-GCA trna GluE2 NNN-CGA trna GluE2 NNN-GGA trna GluE2 NNN-CUA trna Lys CUU-NNA trna Lys CUU-NNA trna Lys CUU-NNA trna Lys CUU-NNA trna Lys CUU-CCA trna Lys CUU-CGA trna Asp GUC-NNA trna Asp GUC-NNA trna Asp GUC-NNA trna Asp GUC-NNA trna Asp GUC-CCA trna Tyr GUA-NNA trna Tyr GUA-NNA trna Tyr GUA-NNA trna Tyr GUA-NNA trna Tyr GUA-CCA trna AsnE2 NNN-NNA trna AsnE2 NNN-NNA trna AsnE2 CUU-NNA trna AsnE2 GUA-NNA trna AsnE2 GUC-NNA trna AsnE2 NNN-CCA trna AsnE2 NNN-GCA trna AsnE2 NNN-CGA trna AsnE2 NNN-GGA trna AsnE2 NNN-CUA DNA template DNA template DNA template (Heptapeptide-1) 23

24 Sequence(5' to 3') used for GTACTTGTCGTACTTCATGTTTTTCTCCTTATTAAAGCC DNA template (Heptapeptide-2, 3) GTCCTTCTTGTCCTTCATGTTTTTCTCCTTATTAAAGCC DNA template (Heptapeptide-4, 5) CGAAGCTCACTTGTACTTCTTGTACTTCATGTTTTTC DNA template (Heptapeptide-1) CGAAGCTCAGTCGTACTTGTCGTACTTCATGTTTTTC DNA template (Heptapeptide-2, 3) CGAAGCTCACTTGTCCTTCTTGTCCTTCATGTTTTTC DNA template (Heptapeptide-4, 5) GGTGGGTAGTTTGACTGCGGCGGTCTCCTCCTAAAGAG CTCTTTAGGAGGAGACCGCCGCAGTCAAACTACCCACC GGTGGGTAGTTTGACTGGCGCGGTCTCCTCCTAAAGAG CTCTTTAGGAGGAGACCGCGCCAGTCAAACTACCCACC GGTGGGTAGTTTGACTGCCGCGGTCTCCTCCTAAAGAG CTCTTTAGGAGGAGACCGCGGCAGTCAAACTACCCACC GGTGGGTAGTTTGACTGAGGCGGTCTCCTCCTAAAGAG CTCTTTAGGAGGAGACCGCCTCAGTCAAACTACCCACC GTCCCAAGGGTATGGCTCTTCGCCATTTAAAGTGGTAC GTACCACTTTAAATGGCGAAGAGCCATACCCTTGGGAC GTCCCAAGGGTATGGCTATTCGCCATTTAAAGTGGTAC GTACCACTTTAAATGGCGAATAGCCATACCCTTGGGAC Quick Change (G2251C) Quick Change (G2251C) Quick Change (G2252C) Quick Change (G2252C) Quick Change (G2251C/G2252C) Quick Change (G2251C/G2252C) Quick Change (G2251A) Quick Change (G2251A) Quick Change (G2553C) Quick Change (G2553C) Quick Change (G2553A) Quick Change (G2553A) TACTCTTTAGGAGGAGACCG Primer extension (2251, 2252) GCGTACCACTTTAAATGGCG Primer extension (2553) CCACCCTTTAATGTTTGATGTTC Sequencing (2251, 2252) CAGTCAAGCTGGCTTATGC Sequencing (2251, 2252) CATATCGACGGCGGTGTTTG Sequencing (2553) GGCAGATAGGGACCGAAC Sequencing (2553) N(M) indicates 2 -O-methylated nucleotide. 24

25 References for Supplementary Information 26 Kang, T. J. & Suga, H. FEBS Lett. 585, (2011). 27 Nissen, P. et al. Science 270, (1995). 28 Liu, J. C., Liu, M. & Horowitz, J. RNA 4, (1998). 29 Schmeing, T. M. et al. Science 326, (2009). 25

TRANSLATION. Translation is a process where proteins are made by the ribosomes on the mrna strand.

TRANSLATION. Translation is a process where proteins are made by the ribosomes on the mrna strand. TRANSLATION Dr. Mahesha H B, Yuvaraja s College, University of Mysore, Mysuru. Translation is a process where proteins are made by the ribosomes on the mrna strand. Or The process in the ribosomes of a