Figure 5.

yDcpS decapping of a 25mer Cap1 RNA followed by VCE recapping. A m⁷G-capped 25mer RNA (0.22 μg) was mixed with 1 µg of total E. coli RNA and incubated with 0.1 nmol of yDcpS for 2 h at 37 °C. An aliquot was removed prior to the addition of yDcpS to provide a reference band for m⁷G-capped 25mer (Lane 1). The yDcpS reaction was terminated by addition of Proteinase K and purified using Ampure XP beads. After elution, the RNA was incubated in 1X VCE buffer containing 0.1 mM SAM and 0.5 mM DTB-GTP in the presence (Lane 2) or absence (Lane 3) of VCE for 1 h at 37 °C. All samples were electrophoresed on a 15% TBE-Urea gel and stained with SYBR Gold.