Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation
Figure 5
The RNAIII-coa mRNA complex prevents ribosome binding and promotes RNase III cleavages.
(A) Formation of the ternary complex between coa mRNA (15 nM), S. aureus 30S ribosomal subunits (250 nM), and initiator tRNA (1 µM) was monitored in the absence (lane 3) or in the presence of increasing concentrations of wild-type RNAIII, RNAIII-Δ7–9 (Δ7–9), RNAIII-Δ13 (Δ13), and RNAIII-Δ14 (Δ14): lane 4, 25 nM; lane 5, 50 nM; lane 6, 100 nM. The toeprint at position +16 is indicated. Lanes 1, 2: Incubation controls on free RNA or RNA bound to RNAIII, respectively. Lanes U, G: dideoxy-sequencing reactions performed on coa mRNA. (B) RNase III hydrolysis of 5′-end-labeled coa mRNA, alone (lane 3) or in the presence of an excess of wild type RNAIII, RNAIIIΔ7–9 (Δ7–9), RNAIII-Δ13 (Δ13), RNAIII-Δ14 (Δ14): lane 4, 25 nM; lane 5, 50 nM; lane 6, 100 nM. Lanes 1, 2: incubation controls on free coa mRNA or bound to RNAIII, respectively. Lanes T1, L: RNase T1 and alkaline ladders, respectively. (C) RNase III hydrolysis of 5′-end-labeled coa mRNA, alone (lane 3) or in the presence of an excess of wild type RNAIII (lane 4), the 3′ domain of RNAIII (3′ Dom), and the hairpin 7 of RNAIII (H7). Lanes T1, L: RNase T1 and alkaline ladders, respectively.