Binding of the RNase III
dsRBD to R1.1[WC] RNA is resistant to EB. A gel
shift assay was performed using 5′-32P-labeled
R1.1[WC] RNA and purified dsRBD, as described
in Materials and Methods. We have shown elsewhere that the RNase
III dsRBD binds R1.1[WC] RNA with a Kd of ∼800 nM (A.K.Amarasinghe, S.Su, W.Sun,
R.W.Simons and A.W.Nicholson, manuscript in preparation). CaCl2 (10
mM) was included in the binding reaction and gel and running buffers. The
reaction was analyzed by phosphorimaging. The dsRBD concentration
was 800 nM. The positions of bound and free R1.1[WC] RNA
are indicated. The smear of radioactivity reflects dissociation
of the dsRBD–R1.1[WC] RNA complex during
electrophoresis. Lane 1, no dsRBD; lane 2, no EB; lane 3, 4 µM EB; lane 4, 12 µM
EB; lane 5, 20 µM EB; lane 6, 40 µM EB; lane 7, 100 µM
EB.