Fig. 5.

Gel-mobility shift assays demonstrating RNA-binding activity of recombinant WTF1 and DUF860. The proteins indicated at top were incubated with (A) a 141-nt RNA from the petB intron, or (B) a 31-nt RNA from the petA gene. The latter RNA was either heated and snap-cooled to maintain it in single-stranded form (at left, SS), or annealed to its complement (at right, DS). Proteins were used in serial 2-fold dilutions (maximum concentrations of 1 μM for MBP and MBP-WTF1, and 2 μM for MBP-DUF860). RNAs were present at 20 pM. Bound (B) and unbound (U) RNAs were resolved on native polyacrylamide gels. (C) Purity of MBP-DUF860 used for RNA-binding assays. Consecutive fractions from the gel filtration column used as the final purification were analyzed by SDS/PAGE and staining with Coomassie Blue. The purity of MBP-WTF1 is shown in Fig. 6.