Figure 4
From: The yfhQ gene of Escherichia coli encodes a tRNA:Cm32/Um32 methyltransferase
Nuclease P1 cleavage of in vitro transcribed E. coli tRNASer1 and tRNAGln2 incubated with YfhQ and AdoMet. Autoradiography of two-dimensional chromatograms of 5'-phosphate and 3'-phosphate nucleosides on thin layer cellulose plates. [α-32P]CTP-labelled in vitro transcribed tRNASer1 (2.5 × 105 cpm) (a and c) and [α-32P]UTP-labelled in vitro transcribed tRNAGln2 (2.5 × 105 cpm) (b and d) was incubated in the presence (a and b) or absence (c and d) of the YfhQ protein. The reaction mixture contained 50 mM PIPES-Na, pH 7.0, 4 mM MgCl2, 50 μM AdoMet and 5 μg of the purified YfhQ protein. After 90 min incubation at 37°C, the tRNA was recovered, cleaved by nuclease P1, the resulting nucleotides were analyzed as described [34]. The additional spot marked with an asterisk ("*") corresponds to an unknown species, a product either of an unusual cleavage pattern modified by the presence of 2'-O-methylation or degradation.