Figure 2.

RlhA/YdcP is responsible for ho⁵C2501 formation. (A) RNA-MS of 48-mer segments of 23S rRNA from WT (leftmost panels), ME5100 (left panels), ΔydcP (right panels) and ME5100 rescued by plasmid-encoded ydcP (rightmost panels). Shown are extracted mass chromatograms (XICs) for divalent negative ions of the RNase T₁–digested heptamer fragments with (upper panels) or without (lower panels) ho⁵C2501. Frequencies of ho⁵C2501 are indicated. n.d., not detected. (B) Collision-induced dissociation (CID) spectra of the heptamer fragments with (left panel) or without (right panel) ho⁵C2501. A divalent ion for each fragment (m/z 1123.1 and 1115.1) was employed as the parent ion for CID. c, y, and w series product ions are assigned in each sequence. (C) E. coli genomic region from ycjD to ydfJ, and a series of deletion strains used to narrow down the gene responsible for ho⁵C2501 formation. The white bar represents a deleted region. The presence (+) or absence (–) of ho⁵C2501 is denoted in each strain. (D) Sucrose density gradient profiling of SPA-tagged RlhA in ribosomal fractions. Upper panel shows UV traces at 260 nm for the ribosomal fractions in 0.5 (filled circles) and 10 mM (open circles) Mg²⁺ conditions. Lower panels show western blotting to detect SPA-tagged RlhA protein in each Mg²⁺ concentration.