Table 1.

Assay conditions for in vitro conversion of ms²i⁶A to ms²io⁶A catalyzed by MiaE2H

Experiments	tRNA	MiaE2H	Cell-free extracts	Dialyzed cell-free extracts	NADPH	H2O2	ms2io6A
1	+		+				−
2	+	+					−
3	+	+	+				+
4	+	+		+			−
5	+	+		+	+		+
6	+	+				+	+

The hydroxylase activity was assayed by incubating ≈50–100 μg of tRNAs with (+) or without (blank) 50 μM purified MiaE2H and 30 μl of cell-free extracts at 20 mg/ml in aerated buffer (Experiments 1–3). In Experiments 4 and 5, the cell-free extracts were dialyzed and assayed without (Experiment 4) or with (Experiment 5) 1 mM NADPH. In Experiment 6, 20 mM H₂O₂ was used in place of cell-free extracts. The last column indicates whether the experiment resulted (+) or not (−) in the production of ms2io6A as a probe for tRNA hydroxylation.