(A) Nucleoside analysis of tRNA fractions from T. thermophilus wild-type (WT) and ΔthiI strains. Absorbances at 254 nm (upper) and 330 nm (lower) were monitored. Transfer RNA fraction from the wild-type strain contains s4U and m5s2U. In contrast, the tRNA fraction from the ΔthiI strain contains only m5s2U. The peak areas of s4U and m5s2U in the lower panels do not represent the molecular ratio between s4U and m5s2U, because the absorption of UV at 330 nm by m5s2U is weak. (B) CD-spectra of tRNA fractions from T. thermophilus wild-type (WT) and ΔthiI strains. The presence of m5s2U in the tRNA fraction from the ΔthiI strain was confirmed. (C) Transfer RNA fractions from T. thermophilus wild-type and ΔthiI strains reacted with MTSEA biotin-XX were separated by 10% PAGE (7 M urea) (left) and visualized by methylene blue staining. The RNAs were blotted onto a nylon membrane and then chemiluminescence derived from bound streptavidin-HRP was monitored.
