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Figure 2. Status of rRNA methylations upon SAM depletion

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1 Figure 2. Status of rRNA methylations upon SAM depletion
Figure 2. Status of rRNA methylations upon SAM depletion. (A) MS analyses of m5U1407 (upper panels) and ... Figure 2. Status of rRNA methylations upon SAM depletion. (A) MS analyses of m<sup>5</sup>U1407 (upper panels) and m<sup>3</sup>U1498 (lower panels) in 16S rRNA from WT 30S (left panels) and Δmtn 30S (right panels). Extracted ion chromatograms (XICs) of the indicated negative ions of the RNase T<sub>1</sub>-digested fragments with (red lines) or without (black lines) target methylation are shown. (B) MS analyses of four methylations in 23S rRNA from WT 50S (left panels), Δmtn 45S (center panels) and Δmtn 50S (right panels). XICs of the indicated negative ions of the RNase T<sub>1</sub>-digested fragments with (red lines) or without (black lines) target methylation are shown. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, gkz111, The content of this slide may be subject to copyright: please see the slide notes for details.

2 Figure 1. The ribosomal 45S precursor accumulates in Δmtn strain
Figure 1. The ribosomal 45S precursor accumulates in Δmtn strain. (A) Growth properties of WT and Δmtn strains. (Left) ... Figure 1. The ribosomal 45S precursor accumulates in Δmtn strain. (A) Growth properties of WT and Δmtn strains. (Left) Overnight full-growth cultures were serially diluted (OD<sub>600</sub> from 10<sup>−1</sup> to 10<sup>−6</sup>), spotted onto LB plates and cultivated at 37, 30 or 22°C. (Right) The doubling time at each temperature was measured. Data represent average values ± SD from biological triplicates. Asterisks indicate the P-value from two-tailed t-tests (*P < 0.01, **P < 0.001). (B) Growth curves of WT (open squares), Δmtn (closed circles), and the Δmtn strain complemented with plasmid-encoded mtn (open circles) cultured at 22°C in LB medium. (C) Sucrose density gradient (SDG) profiles of ribosomes from WT, Δmtn and ΔrlmE strains performed at low Mg<sup>2+</sup> concentration (0.5 mM) and high Mg<sup>2+</sup> concentration (10 mM). All strains were cultured at 22°C in LB medium. The y-axis represents milli-absorbance units at 260 nm. (D) Ribosomal proteins of the 45S precursor and the 50S subunit from WT, ΔrlmE and Δmtn strains resolved by Tricine-SDS-PAGE. The gel was stained with SYPRO Ruby. L36 is indicated by an arrow. All strains were cultured at 22°C in LB medium. (E) Absorbance ratio of 30S (gray squares), 45S (open circles) and 50S (closed circles) species relative to total ribosomes at various temperatures. Ratios were calculated from the peak area in the SDG profiles described in Supplementary Figure S3. (F) Growth curves of WT (open circle) and Δmtn (close circle) strains cultured at 30°C in M9 medium supplemented with 18 amino acids (without Met and Cys) (Left panel) or 19 amino acids (without Cys) (Right panel). (G) SDG profiles of ribosomes from the Δmtn strain cultured at 30°C in M9 medium supplemented with 18 amino acids (without Met and Cys) (Left panel) or 19 amino acids (without Cys) (Right panel). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, gkz111, The content of this slide may be subject to copyright: please see the slide notes for details.

3 Figure 3. Status of tRNA methylations upon SAM depletion
Figure 3. Status of tRNA methylations upon SAM depletion. (A) The relative abundance of tRNA modifications in the Δmtn ... Figure 3. Status of tRNA methylations upon SAM depletion. (A) The relative abundance of tRNA modifications in the Δmtn strain (light gray) versus the WT strain (dark gray) were measured by MS analysis of nucleosides. All modifications except for m<sup>1</sup>G and Gm were analyzed by HILIC/ESI-MS, while m<sup>1</sup>G and Gm were analyzed by RPC/ESI-MS using a polymer-coated core shell column containing resin with adamantyl groups (see ‘Materials and Methods’ section). The relative abundance of each nucleoside was normalized against the respective unmodified nucleoside. Data represent average values ± SD from biological triplicates. Asterisks indicate modifications for which the frequency was statistically decreased in the Δmtn strain (*P < 0.025, **P < 0.01, ***P < 0.001). (B) XICs of the proton adducts of nucleosides for ms<sup>2</sup>i<sup>6</sup>A and i<sup>6</sup>A in WT and Δmtn strains. Asterisks represent unrelated fragments with the same m/z values. Frequency of ms<sup>2</sup>i<sup>6</sup>A was measured from the proportion of the ms<sup>2</sup>i<sup>6</sup>A peak area to the total peak areas of ms<sup>2</sup>i<sup>6</sup>A and i<sup>6</sup>A, because the ionization efficiencies of these nucleosides were almost identical in our measurement (37). (C) XICs of the proton adducts of nucleosides for queuosine and its precursors in WT and Δmtn strains. The relative abundance was normalized by the abundance of guanosine. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, gkz111, The content of this slide may be subject to copyright: please see the slide notes for details.

4 Figure 4. Overexpression of rlmE restores the growth phenotype of the Δmtn strain. (A) Doubling time of WT and Δmtn ... Figure 4. Overexpression of rlmE restores the growth phenotype of the Δmtn strain. (A) Doubling time of WT and Δmtn strains overexpressing different RNA-modifying enzymes. All strains were cultured at 22°C. Data represent average values ± SD from biological triplicates. Asterisks indicate P-values from two-tailed t-tests. (B) Growth curves of the Δmtn strain overexpressing a mock gene (closed circles) and rlmE (open circles) cultured at 22°C in LB medium. (C) SDG profiles of ribosomes from Δmtn strains overexpressing different RNA-modifying enzymes as indicated. All strains were cultured at 22°C. (D) Mass spectrometric analyses of Um2552 in 23S rRNAs in the 45S precursor (left panel) and 50S subunit (right panel) from the Δmtn strain overexpressing rlmE. XICs of the indicated negative ions of the RNase T<sub>1</sub>-digested fragments with (red lines) or without (black lines) Um2552. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, gkz111, The content of this slide may be subject to copyright: please see the slide notes for details.

5 Figure 5. The regulatory role of SAM in 50S assembly
Figure 5. The regulatory role of SAM in 50S assembly. (A) Mechanistic model for late assembly of 50S subunits triggered ... Figure 5. The regulatory role of SAM in 50S assembly. (A) Mechanistic model for late assembly of 50S subunits triggered by Um2552 formation. The 45S precursor is a flexible molecule that lacks Um2552 and several r-proteins including L36. RlmE-mediated Um2552 formation promotes the interaction of helices H92 and H71, and incorporation of L36 and other r-proteins. This process triggers 50S maturation in the peptidyl transferase center (PTC) and central protuberance (CP). The chemical scheme of Um2552 formation is shown in the lower figure. SAH, S-adenosyl-L-homocysteine. (B) Regulatory mechanism of 50S assembly mediated by Um2552 modification via sensing of the intracellular SAM concentration. When SAM is abundant, RlmE introduces Um2552 into the 45S precursor, which promotes 50S assembly and thereby maintains efficient translation. When SAM is less abundant, Um2552 remains hypomodified and the 45S precursor accumulates, leading to less efficient translation. As the 45S precursor is stably present in cells, 50S subunits can be generated when SAM levels in the cell recover. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Nucleic Acids Res, gkz111, The content of this slide may be subject to copyright: please see the slide notes for details.

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