Figure 6

Complete RNAse T1 cleavage followed by nuclease T2 cleavage of in vitro transcribed E. coli tRNA^Ser1 incubated with YfhQ and AdoMet. Autoradiography of a two-dimensional chromatogram of 5'-phosphate nucleosides on a thin layer cellulose plate. [α-32P]CTP-labelled in vitro transcribed tRNA^Ser1 (1.5 × 10⁶ cpm) was incubated with 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, digested overnight at 37°C by 400 u RNAse T1 in 100 mM Tris-HCl pH 7.5. The resulting oligonucleotides were separated on a 20% polyacrylamide gel (19:1) and revealed by autoradiography. Oligonucleotides were eluted from the gel and cleaved by nuclease T2. The resulting nucleotides were analyzed as described [38]. This image shows the TLC analysis for a 5-nucleotide long fragment (nucleotides 31–35).