Fig. 5.

Stoichiometry and affinity between p20 and TAR by native PAGE. Native gel assays were performed using electrophoretically unique variants of p20 and TAR RNA. For p20, a VP16 peptide was fused to the C-terminus (p20-VP16), while 30 adenosines were fused to the 3’-end of TAR (TAR-A30). All RNAs were renatured by incubating at 90 °C to populate the monomer. Trace (~2 nM) 5’-end labeled RNA was incubated with 1 µM p20, 4 µM p20-VP16, or both proteins for 1 h at room temperature. Native gels are 10% polyacrylamide, 0.5×TBE, and were run at 4 °C. (a) p20, p20-VP16, or both were incubated with TAR and fractionated by native PAGE. Free TAR was also run on the gel. Data are consistent with a 1:1 complex. (b) TAR, TAR-A30, or both were incubated p20 or alone, and fractionated by native PAGE. Data are consistent with a 1:1 complex. (c) Truncated TAR variants, shTAR51, shTAR47, and shTAR43, were tested for binding to p20. Data show that all three truncated TARs bind p20, but less strongly as RNA length decreases. Percent RNA shifted is provided below the gel.