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. 2019 Nov 15;48(1):359–372. doi: 10.1093/nar/gkz1065

Figure 3.

Figure 3.

Methylation of 30S subunits is important for RbfA release. (A) Methylation of 16S A1519 in pre-30S and 30S subunits was measured by primer extension (see Materials and Methods). Pre-30S subunits from BX41 (ΔrbfA) were methylated by KsgA (lane 4); nm30S subunits from ΔksgA cells (JW0050-3) and TPR201 cells bearing an inactive ksgA allele were unmethylated (lanes 6 and 7). (B) Pelleting assay showing that neither IF3 (top) nor RsgA (bottom) promote Cy5-RbfA release from nm30SΔksgA subunits and promote only partial release from nm30STPR201 subunits. (C) % Bound RbfA relative to buffer control as in panel B; mean and s.d.; n = 2. (D) Native PAGE showing that Cy5-RbfA and Cy3-IF3 can bind pre-30S or 30S subunits simultaneously. 30S complexes were purified from the strains shown and incubated with Cy5-RbfA and Cy3-IF3 before native PAGE. Panels show the same gel scanned with Cy3 excitation (bottom), Cy5 excitation (middle), and FRET to Cy5 upon Cy3 excitation (top). The FRET efficiencies are indicated at the bottom.