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. 2015 Jun;21(6):1203–1216. doi: 10.1261/rna.049171.114

FIGURE 3.

FIGURE 3.

YjeQ M3 is unable to remove RbfA bound to the mature 30S subunit. (A) Filtration assay to test the release of RbfA (R) from the mature 30S subunit (Mt 30S) in the presence of YjeQ wild-type (Y) and YjeQ M3 variant (M3). These reactions were performed by adding the assembly factors in fivefold molar excess with respect to the 30S subunits and in the presence of GMP-PNP. Incubations were done at 25°C. Flow-through (FT) and bound (B) fractions from the filtration assay were resolved in 4%–12% bis–tris SDS-PAGE and stained with Coomassie blue. (B) Pelleting assay of identical reactions used in A. The molecular weight marker (M) is in kDa. Supernatant (S) and pellet (P) fractions from this assay were resolved in 4%–12% bis–tris SDS-PAGE and stained with Coomassie blue. The bar diagrams under the gels indicate the binding of the RbfA to the mature 30S subunit in each reaction. The observed binding of RbfA to the mature 30S subunit was defined as 1.