d Yield of peptide (µm)

Transcription

1 a Pro template DN native trn mix pre-charged Pro-tRN Pro T7 RN polymerase [ 4 ]sp Met Lys ly sprs MetRS LysRS lyrs E. coli ribosome Tyr TyrRS 5 amino acids and 5 aminoacyl-trn synthetases other protein factors b mr P mr2 P2 mr3 P3 U U U U U fm K K K P D Y K D D D D K stop U U U U fm K K K P P D Y K D D D D K stop U U U U fm K K K P P P D Y K D D D D K stop c peptide [ 4 ]sp EF-P mrn mr mr2 mr3 mr2 peptide P P2 P3 P2 Pro-tRN Pro d 3 2 mrn peptide P P2 P3 N.D. N.D. N.D. N.D. mr mr2 mr3 mr2 Pro-tRN Pro P2 Supplementary Figure EF-P effect on the synthesis of Pro-containing sequences in the FIT system. (a) Schematic depiction of peptide translation in the FIT system. For incorporation of Pro, in vitro transcribed E. coli trn Pro was pre-charged with Pro by means of the flexizyme technology and added to the translation system. Other amino acids were charged on natural E. coli trns de novo by the corresponding aminoacyl-trn synthetases (RSs) present in the FIT system. [ 4 ]sp was used for autoradiographic detection of peptides. (b) Sequences of mrns (mr 3) and the corresponding peptide sequences (P 3). These mrns were transcribed from the corresponding DN templates by T7 RN polymerase present in the FIT system. (c) Tricine SDS-PE analysis of the peptides (P-P3) synthesized in the FIT system. Translation was carried out for 2 min at 37 in a 2.5-µL reaction mixture in the presence (3 µm) or absence of EF-P. (d) Yields of the full-length peptides (P 3) synthesized by the FIT system. The [ 4 ]sp-labeled peptide in the tricine SDS-PE gel was quantified by autoradiography. Numbers above the bars indicate the relative translation yields of the peptides calculated by [EF-P(+)/EF-P( )]. N.D.: not detected. Error bars, s.d. (n = 3).

2 a trn Pro -WT/EF-P(+) trn Pro -WT/EF-P(-) b.5 trn Pro -3/EF-P(+) trn Pro -3/EF-P(-).5 trn Pro -WT trn Pro Time (min). 5 5 EF-P conc. (µm) Supplementary Figure 2 Time-course analysis and titration of EF-P concentration in translation. (a) Time-course analysis in translation of P2-Pro 2 (Fig. 2a) using wildtype (WT) trn Pro and 3 mutant in the presence (3 µm) and absence of EF-P. Error bars, s.d. (n = 3). (b) Titration of EF-P concentration in P2-Pro 2 (Fig. 2a) translation using the wildtype (WT) trn Pro and 3 mutant for Pro incorporation. Translation time is 2 min. Error bars, s.d. (n = 3).

3 mrn mr2 mr2 mr2 mrn mr2 mr4 mr5 mr6 peptide P2 P2 P2 peptide P2 P4 P5 P6 Xaa Pro Me la Me Thr a codon (XXX) U U UU UU U U codon (XXX) U U trn body Ser4 Trp Leu2 Ile la2 Pro Pro2 Pro3 trn body Ser4 Pro Ser4 Pro anticodon U U anticodon c d e f trn chim trn Pro (WT) g b.3.2. mrn mr4 mr4 mr4 mr4 peptide P4 P4 P4 P4 Pro-tRN Pro Pro-tRN Ser Me Thr-tRN Pro Me Thr-tRN Ser U 7aU U7b 8U U2 2 WT trn Pro Supplementary Figure 3 Quantification of absolute translation yields of peptides. (a-n) bsolute translation yields of the peptides obtained in the experiments of Fig. 2b (a), Fig. 2c (b), Fig. 3b (c), Fig. 3d (d), Fig. 3e (e), Fig.4b (f), Fig. 4c (g), Fig. 4d (h), Fig.4e (i), Fig. 4g (j), Fig. 5b (k), Fig. 5d (l), Fig. 5f (m) and Fig. 6b (n). Translation products of each experiment were separated by 5% tricine SDS-PE, and the corresponding peptide labeled with [ 4 ]sp were quantified by autoradiography. Black and white bars indicate results of translation with and without EF-P, respectively. Error bars, s.d. (n = 3). (o) Yields of f[ 35 S]Met-puromycin obtained in the experiment of Fig. 6d were analyzed by liquid scintillation counting. Black and white bars indicate results of reaction with and without EF-P, respectively. Error bars, s.d. (n = 3).

4 h U U U U3 3 3 U i.5.5 U U U U U j a +7cU WT (8 nt) ( nt) (9 nt) k WT 2 3 trn Pro trn Pro trn Ser4 l WT trn Pro m WT 2 trn Pro trn la2 trn Val2 n WT trn Pro o Peptide/7S (%) WT trn fmet trn fmet U Supplementary Figure 3 (continued)

5 a Relative translation yield, EF-P(+)/ EF-P(-) U 3 U2 6U 7aU 3 3 6U 7aU U2 6U 7aU 7aU 7aU U2 U2 WT trn Pro b U U s 4 U U U U U U T ψ U D m 7 U U Um U m c Relative translation yield, EF-P(+)/ EF-P(-) 3 2 trn transcript native native trn Pro Supplementary Figure 4 Effects of multiple point mutations and nucleotide modifications. (a) Effect of double or triple point mutations introduced into trn Pro on incorporation of Pro. Relative translation yields were analyzed using mr2-2 mrn (Fig. 2a). Error bars, s.d. (n = 3). (b) Secondary structure of native trn Pro with nucleotide modifications indicated by red. s 4 U: 4-thiouridine, D: dihydrouridine, Um: 2'-O-methyluridine, m : -methylguanosine, m 7 : 7-methylguanosine, T: ribothymidine, ψ: pseudouridine (c) Incorporation of [ 4 ]Pro at tandem codons of mr2-2 mrn using transcript and native trn Pro/Pro2/Pro3 mixture. Values represent relative translation yields calculated as [EF-P(+)/EF-P( )]. Error bars, s.d. (n = 3).

6 a mrn FLK-FL U U U UU U U U U U fm I Q P I S P P P Q P P U U U U U Q D N L D Y K D D D D K stop b Relative translation yield, EF-P(+)/ EF-P(-) WT 3 trn Pro/Pro2/Pro3 Supplementary Figure 5 Improvement of translation of FLK-FL by EF-P depending on D-arm structure of trn Pro used for Pro incorporation. (a) Sequence of mrn and FLK-FL peptide, 2 amino acids from the N terminus of FLK protein followed by FL sequence. (b) Improvement of translation yield of FLK-FL by EF-P. Pro was pre-charged on wildtype trn Pro/Pro2/Pro3 mixture or their D-arm mutants (3), and used for Pro incorporation in translation of FLK-FL. Translation product was separated by 5% tricine SDS-PE, and the desired peptide labeled with [ 4 ]sp was quantified by autoradiography. Values represent relative translation yields calculated as [EF-P(+)/EF-P( )]. Error bars, s.d. (n = 3).

7 Supplementary Figure 6 Time courses of peptide bond formation between fmet-pro and ly using D-arm variants of trn Pro and trn Ser4. The reaction was initiated by mixing posttranslocation complex containing [ 4 ]fmet-pro-trn and [ 3 H]ly-tRN ly3 EF-Tu TP ternary complex, and reaction products were analyzed by HPL. Posttranslocation complexes were prepared with the 6 different Pro-tRN variants shown in Fig. 6a,b.

8 Supplementary Figure 7 Mass spectrometric analysis of EF-P modification. (a) Mass spectra of the unmodified (z=3; m/z= (monoisotopic ion)) and modified peptides (z=4; lysylated m/z= ; lysylated/hydroxylated m/z= (monoisotopic ions)). Series of other isotopic ions are indicated as [M+~5]. (b) Library MS/MS spectra of the unmodified (z=3) and modified peptides (z=4). The modified lysine residue is indicated in red in the peptide sequence. (c) Elution profile of parallel reaction monitoring transitions (PRM). Peptides were identified by co-elution of their PRM transitions and comparison to the reference library spectrum (dot product). Example PRM profiles show the modification status of EF-P used in this work. (d) Relative label-free quantification of the EF-P modification status by PRM. Bars represent the sum of 7 PRM transitions. Error bars indicate standard deviations of three independent technical replicates.

9 Supplementary Table omparison of the D-arm sequences of E. coli trns. Watson-rick base-pairing (/U and /) and wobble base-pairing (/U) nucleotides are shown in red and blue, respectively. /U wobble base pairs are also considered as base pairs for counting the length of the D-loop and stem region.

10 Supplementary Table 2 omparison of the D-arm sequences among different organisms. Watson-rick base-pairing (/U and /) and wobble base-pairing (/U) nucleotides are shown in red and blue, respectively. /U wobble base pairs are also considered as base pairs for counting the length of the D-loop and stem.