Figure 1.
Overexpressed tRNAGlu from a ΔMnmC E. coli strain. (a) HPLC of total extracted tRNA. HPLC buffers: 100 mM Tris pH 8, 50 → 150 mM MgCl2. The four major modivariants are labelled 1–4. Each was isolated by HPLC for subsequent MS analysis of modified nucleosides. (b) Representative purified tRNA (tRNA 5, see below) after anion-exchange purifications at pH 5 and 8. HPLC buffers: 100 mM Tris pH 8, 50 mM MgCl2, 0 → 500 mM NaCl. [Note: The MgCl2 and NaCl gradients in (a) and (b) were used interchangeably for analytical purposes] (c) Diagrams showing the modified nucleosides present in tRNAs 1–5 and those present in tRNAs 5 and 6, the products of reaction of tRNA 1 with MnmC (see below). Nucleosides labelled in red (or orange or yellow) were identified by MS (Figure 2 and Supplementary Figure S1). The presence of 
nucleosides labelled in grey was not confirmed. tRNA 1 contains cmnm5s2U34 and all other expected modifications, while tRNA 2–4 are less fully modified at anticodon–stem loop postions as shown. The nm5s2U and mnm5s2U modifications obtained by reaction with MnmC are shown in orange and yellow, respectively, to differentiate from cmnm5s2U.