Figure 3.

Mutations in the AdoMet-binding motif I of Gcd14p eliminate m¹A synthesis in vivo. (A) Transformants of yeast strain F516 (gcd14–2) containing low-copy URA3 plasmids bearing wild-type GCD14 (p2599), GCD14Flag (pJA148), gcd14–3Flag (pJA149), or vector alone, were streaked on SC medium lacking uracil and incubated at 36°C for 2 days. (B) HPLC chromatographs of total tRNA isolated from yeast strain yJA203 (gcd14Δ hcIMT4) harboring pJA148 (GCD14Flag) or pJA149 (gcd14–3Flag). The A₂₅₄ was plotted against the time of elution. The identities of known nucleosides or their modified counterparts are indicated above or within the corresponding peaks (Ψ, pseudouridine; C, cytidine; U, uridine; m¹A; m⁵C, 5-methylcytidine).