Figure 3. Biochemical characterization of CMTr1 and CMTr2 and their fragments.

The analysis was performed for full-length proteins and deletion variants of CMTr1 (a,b) and CMTr2 (c,d). The proteins were overexpressed in and isolated from HEK 293 cells (white bars) or E. coli (grey bar). Protein variant CMTr1 126–550 was expressed from crystallization construct. (a,c) MTase activity. In vitro transcribed RNA-GG molecules with a ³²P-labelled cap0 (a) or cap01 (c) structure were incubated with the indicated enzymes in the presence of SAM. Product RNA was digested with nuclease P1 (a) or RNase T2 (c) and purified by phenol/chloroform extraction and ethanol precipitation. The digestion products were resolved on 21% polyacrylamide/8 M urea gel and quantified after autoradiographic visualization. (b,d) Substrate binding. In vitro transcribed RNA-GG molecules with a ³²P-labelled cap0 (b) or cap01 (d) structure were incubated with the indicated enzymes in the presence of SAH (the product of SAM demethylation) and uncapped, competitor RNA to detect specific substrate binding. After 30 min incubation, the samples were filtered through a nitrocellulose membrane and washed with reaction buffer. RNA bound to membrane-attached proteins was visualized by autoradiography and quantified. The signal from the negative control (that is, the sample with BAP protein) was subtracted from the signal from samples with cap MTases. The analyses were performed in triplicate. The relative activity/binding compared with the full-length enzyme (set at 100%) and s.d. values are shown.