Bacteriophage ms2 genomic rna

RNase activity of RnlA and its mutants was monitored by following the cleavage of bacteriophage MS2 genomic RNA (Roche Diagnostics) via denaturing gel electrophoresis as described (28 (link)). RNase-free purifications of all proteins were completed before carrying out the activity assays, by preparing buffers with diethyl pyrocarbonate (DEPC)-pretreated water and/or Ultrapure RNase-free water, using RNase-free laboratory consumables and RNase-cleaned surfaces. Fifty picomoles of RnlA, the reconstituted RnlA:RnlB complex at a 1:2 molar ratio, and 50 pmol of the four alanine substitution RnlA mutants, were all incubated with 0.08 μg μl^–1 of MS2 RNA in 10 μl final volume, in the presence of RNase Inhibitor (SUPERase In™ RNase Inhibitor, Invitrogen). All reactions were incubated for 1 h at 310 K in PBS. The reactions were stopped by addition of 10 μl of 2x loading dye (95% v/v formamide, 0.02% m/V SDS, 0.02% m/V bromophenol blue, 0.01% m/V xylene cyanol, 1 mM EDTA). Samples were loaded after 10 min incubation at 343 K onto 6% urea gels which had been pre-running at 100 V for 20 min in TBE buffer. The low range ssRNA ladder from New England Biolabs was used as a reference in all gels, which were ran at 150 V for 45 min and subsequently stained with ethidium bromide.