Figure 6

E^rns is a nicking endoribonuclease. (a) Schematic representation of the RNase activity assay with the modified hybrids. A single-stranded RNA of positive polarity (ssRNA+) labeled in red together with either a single-stranded methylated RNA (metRNA) or a ssDNA of negative polarity, both labeled in green, were boiled and cooled down at room temperature for hybridization (RNA/metRNA or RNA/DNA). Strep-tag purified wild-type (C171), monomeric (R171) and RNase-inactive mutant (H30F) of E^rns were incubated at the concentrations indicated with 625 nM single-stranded metRNA or DNA (b), double-stranded RNA/DNA− (c) or double-stranded RNA/metRNA-hybrids (d). Samples were separated by 14% SDS-PAGE and fluorescence was analysed with a Li-Cor Odyssey system. Due to the known, defined length of the directly labeled fragments (30 b for ssRNA, 30 bp for dsRNA), no size ladder was applied. Non-cropped gels as representative experiment out of three (b) or four (c,d) are shown.