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.