RNA-binding activities of rTHA8 and rZm-OTP51. (A) Purification of rTHA8 (left) and rZm-OTP51 (right). Proteins were purified from E. coli via successive affinity and size-exclusion chromatography steps, as described in the Materials and Methods. Aliquots of fractions from the size-exclusion column were analyzed by SDS-PAGE; the elution positions of globular size standards are shown below. The bracketed fractions were pooled for a second amylose-affinity step to remove contaminating MBP (THA8) or for direct use in RNA-binding assays (Strep-OTP51). Coomassie-stained gels of the final preparations are shown to the right. Residual MBP remained in the THA8 preparation, but MBP does not bind RNA (Kroeger et al. 2009). (B) Gel mobility-shift assays using rTHA8 (left) and Strep-OTP51 (right). Radiolabeled RNAs of ∼150 nt were derived from regions of the ycf3-2 intron, as diagrammed below. (B) Bound RNA; (U) unbound RNA.
