Supplementary Figure 1

Transcription

1 Supplementary Figure 1 Isolation of mt-trnas and RNA-MS analysis of mt-trna Asn from M. nudus (a)m. nudus mt-trnas were isolated by RCC and resolved by 10% denaturing PAGE. The gel was stained with SYBR Gold (Invitrogen) and visualized using an FLA-7000 image analyzer (Fujifilm). The trnas as indicated by arrows were cut out of the gel and purified. (b) Top panel: base peak chromatogram (BPC) of RNase T 1 -digested fragments; second and third panel: extracted ion chromatograms (XIC) for the negative ions of the anticodon-containing fragments as indicated. Molecular mass of each fragment numbered on the BPC is listed in Supplementary Table 1. Bottom panel shows the CID spectrum of the cyanoethylated anticodon-containing fragment. The c- and y-series product ions are indicated on the CID spectrum and assigned on the corresponding sequence. (c) Secondary structure of M. nudus mt-trna Asn with post-transcriptional modifications. The numbering system of trna is based on the trna database (Juhling, F. et al., Nucleic Acids Res. 37, D159-62, 2009). m 1 A: 1- methyladenosine, m 2,2 G: N 2, N 2 -dimethylguanosine, Ψ: pseudouridine, and t 6 A: N 6 - threonylcarbamoyladenosine.

2 Supplementary Figure 2 RNA-MS analysis of M. nudus mt-trna Lys XICs of the anticodon-containing fragments with N 428 at position 37 (upper panel), t 6 A37 (lower panel). Sequence, m/z values, and charge state of each fragment are indicated on the right side. The frequency of each modification was calculated from the ratio of the peak areas of the two fragments.

3 Supplementary Figure 3 CID spectrum of t 6 A base The t 6 A base was generated by in-source fragmentation of t 6 A nucleoside in individual E. coli trna Thr4. The product ions are assigned on the chemical structure of the t 6 A base. The internal fragment of the threonine moiety is indicated by a dotted line.

4 Supplementary Figure 4 Enzymatic synthesis of Thr and 4-hydroxythreonine LtaE catalyzes an aldol condensation reaction of acetaldehyde and glycine to synthesize Thr utilizing pyridoxal phosphate (PLP) as a cofactor. LtaE catalyzes the same reaction with glycolaldehyde and glycine to form 4- hydroxythreonine.

5 Supplementary Figure 5 RPC-LC/MS co-injection analysis of the synthetic ht 6 A and natural N 428 XICs of synthetic ht 6 A (left panel), nucleosides of M. nudus mt-trna Lys (middle panel), and co-injection of both samples (right panel). Lower panels show XICs of m 1 A (m/z 298) as controls.

6 Supplementary Figure 6 CID analysis of the synthetic ht 6 A CID spectra of ht 6 A nucleoside (upper panel) and its base ion (lower panel). ht 6 A base (BH 2 + ) was generated by in-source fragmentation of ht 6 A nucleoside of E. coli trna Lys transcript bearing ht 6 A37. The product ions are assigned in the corresponding chemical structures. The internal fragment of the methyl threonine moiety is indicated by a dotted line.

7 list of DNA probes for RCC and primers Purpose Name 5' to 3' sequence probes for RCC mt trna Asn mt trna Asn probe AACGGCCAAGCGCCTTTACATTTAGCTACGACCCA mt trna Lys mt trna Lys probe TGGTCCTTAATAATAGGTATTAGCTATTTTCTTTTAAATTAAGAGTTTAA vector construction pet28a LtaE ltae fw AGTCAGTCAGTCCATATGATTGATTTACGCAGTGATAC ltae rv GACTGACTGACTCTCGAGTTAACGCGCCAGGAATG in vitro transcription E.coli trna Lys mutant trna Lys template GGGTCGTTAGCTCAGTTGGTAGAGCAGTTGACTCTTAATCAATTGGTCGCAGGTTCGAATC trna Lys fw GCTAATACGACTCACTATAGGGTCGTTAGCTCAG trna Lys rv TGGTGGGTCGTGCAGGATTCGAACCTGCG E.coli trna Ala2 trna Ala2 template AGCTCAGCTGGGAGAGCGCTTGCATGGCATGCAAGAGGTCAGCGGTTCGATCCCGCTT trna Ala2 fw GCTAATACGACTCACTATAGGGGCTATAGCTCAGCTGGGAGAG trna Ala2 rv TGGTGGAGCTAAGCGGGATCGAACCGC mrna mrna(aaa) template AGGGTTAACTTTAAGTAAGGAGGTACACTGCTAAATAACTGTAGAAAAAA mrna(aag) template AGGGTTAACTTTAAGTAAGGAGGTACACTGCTAAGTAACTGTAGAAAAAA mrna fw GCGAAATTAATACGACTCACTATAGGGTTAACTTTAAG mrna rv TTTTTTCTACAGTTA