Fig. 1.

HPLC chromatograms of tRNA hydrolysates. (A) tRNAs were obtained from E. coli DH5α strain. (B) tRNAs were obtained from an in vivo complementation of DH5α strain transformed with pT₇-miaE2 or pT₇-miaE2H. The identification of ms²i⁶A and ms²io⁶A was based on UV-visible spectra (data not shown) and retention times (ms²i⁶A eluted at ≈60 min and ms²io⁶A at ≈47 min). (C) ms²io⁶A production as a function of reaction time and quantity of purified MiaE2H enzyme (♦, 20 μM; ▴, 5 μM). The assay mixture contained 50–100 μg of bulk tRNAs and 0.6 mg of cell-free extracts in 100 mM Tris·HCl (pH 7.5). Reactions were carried out at 37°C. (Inset) The HPLC detection of ms²i⁶A substrate (elution at 60 min) and ms²io⁶A, product of the reaction (elution at 47 min) the arrows indicate the decrease of the substrate and the increase of the product.