Dimers of the HDV ribozyme activate PKR. (A) Analytical native gel analysis of isolated and purified 1/99 WT monomer and dimer. Gel is 10% native PAGE run in 0.5× TBE. RNA monomers and dimers were first isolated as described in the Materials and Methods section “Purification of RNA monomers and dimers from native gels;” purification gel not shown. Then, certain monomer and dimer samples were added to an equal volume TEN200M20 (indicated with “s” for salt above the gel), followed by incubation at room temperature for 10 min. Prior to adding TEN200M20, certain monomer samples were heat renatured (indicated with “h” for heat above the gel) to remove possible dimer contaminants by incubating at 90°C for 1 min, followed by room temperature for 10 min, then 55°C for 10 min, followed by incubation at room temperature for 10 min. Both monomer-untreated (M) and monomer-treated with heat and salt (M (h+s)) are shown. Percent dimer is provided. This gel confirms that dimer identity has been largely retained and that, most importantly, monomer identity has been retained after native gel purification. (B) PKR activation by isolated and purified 1/99 WT HDV monomer and dimer. A no-RNA lane is provided, and phosphorylation activities are normalized to 0.01 μM 79-bp RNA. Both no-RNA and 79-bp RNA lanes contain 50 mM NaCl and 5 mM MgCl2. The no-RNA lane was subtracted from each lane in order to provide RNA-dependent activation values.
