Filter binding assay
10 nM single-stranded Cy3 labeled M1, trpL RNA or purified 10 nM double-stranded J1uM1 RNA were titrated with PlzA proteins (50 nM, 100 nM, 200 nM, 300 nM, 500 nM, 800 nM, 1000 nM, and 2000 nM) in 1× binding buffer (50 mM Tris-HCl pH 7, 2 mM DTT, 6% glycerol) and incubated at room temperature for 5 min. Nitrocellulose (Whatman PROTRAN BA 83 0.2 µm) and nylon membranes (GE Healthcare positively charged nylon transfer membrane 0.45 µm) were pre-equilibrated in 1× binding buffer and loaded into a slot blot apparatus (Hoefer Scientific Instruments PR 600 slot blot). Wells were washed with 1× binding buffer and pulled through the membranes with a vacuum. 90 µl of a 100-µl reaction was applied to the wells and vacuumed. PlzA-RNA complexes bound to the nitrocellulose membrane and the free RNA flowed through and bound to the nylon membrane. Wells were washed four times with 90 µl 1× binding buffer. RNA was detected using the Cy3 channel on both membranes with a Azure Sapphire imager. RNA bound to each membrane was quantified using ImageJ and the fraction of PlzA bound RNA was calculated as a function of PlzA concentration. At least three replicates were performed for each PlzA protein. Mean curves and standard error of the means were calculated and the data were fit to the Hill equation for multiple binding sites, which yielded dissociation constants.