Figure 7
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 cellular extracts and AdoMet. Autoradiography of two-dimensional chromatograms of 5'-phosphate nucleosides on thin layer cellulose plates. [α-32P]CTP-labelled in vitro transcribed tRNASer1 (2.5 × 105 cpm) (a, b) and [α-32P]UTP-labelled in vitro transcribed tRNAGln2 (2.5 × 105 cpm) (c, d) was incubated with a crude extract of the YfhQ K.O. strain (a and c), or of the MC1061 E. coli strain (b and d). The reaction mixture contained 50 mM PIPES-Na, pH7.0, 4 mM MgCl2, 50 μM AdoMet and 100 μg of total protein. After 90 min incubation at 37°C, the tRNA was recovered, digested by nuclease P1 and the resulting nucleotides were analyzed as described [38].