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. 2009 Mar 14;37(9):2910–2925. doi: 10.1093/nar/gkp158

Figure 6.

Figure 6.

In vitro reconstitution of yW synthesis using recombinant TYW4 with sodium bicarbonate. (A) LC/MS fragment analyses of RNaseT1-digested tRNAPhe with yW-72, treated by recombinant TYW4 in the absence (left panel) or the presence (right panel) of SAM. This graphs show mass chromatographs for anticodon-containing fragments containing yW-72 (m/z 681) (black line) overlaid with mass chromatographs for anticodon-containing fragments containing yW (m/z 691) (red line). (B) LC/MS fragment analyses of RNaseA-digested tRNAPhe with yW-72, treated by recombinant TYW4 in the presence of SAM and NaHCO3. The graphs on the left describe the mass chromatograms shown by the doubly charged ions of anticodon-containing fragments containing yW (GmAAyWAΨp; m/z ≈ 1119). The graphs on the right show the mass spectrum for each anticodon-containing fragment. Upper panels show the LC/MS analysis of tRNAPhe treated with TYW4 and 13C-NaHCO3. Lower panels show the LC/MS analysis of tRNAPhe treated with TYW4 and 12C-NaHCO3. In the right panels, peak ‘a’ is a monoisotopic ion, and peak ‘b’ is an isotopic ion with a mass 1 Da heavier than that of the peak ‘a’. (C) The graphs describing overlaid mass chromatograms, shown by doubly charged ions of anticodon-containing fragments containing yW (m/z 1119.60–1119.75, red line and 1119.0–1119.25, black line). The meaning of the labels ‘a’ and ‘b’ is the same as that in panel B. The theoretical masses of the doubly charged ions of peaks ‘a’ (yW-12C) and ‘b’ (yW-13C) are m/z 1119.188 and m/z 1119.692, respectively. The left panel shows the LC/MS analysis of tRNAPhe treated with TYW4 and 13C-NaHCO3. Thr right panel shows the LC/MS analysis of tRNAPhe treated with TYW4 and 12C-NaHCO3.