Staphylococcus aureus RNAIII Binds to Two Distant Regions of coa mRNA to Arrest Translation and Promote mRNA Degradation
Figure 2
Enzymatic and chemical probing of the structure of the inhibitory RNAIII-coa mRNA complex.
(A) Enzymatic hydrolysis of 5′-end-labeled 3′ domain of RNAIII, alone (lane 3) or in the presence of an excess of coa mRNA (lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM). Lanes 1, 2: incubation controls on free RNA or bound to coa mRNA, respectively. Lanes T, L: RNase T1 under denaturing conditions and alkaline ladders, respectively. T1, V1: RNase T1 and RNase V1 hydrolysis, respectively. (B, C) Enzymatic hydrolysis of 5′-end-labeled coa mRNA, alone (lane 3) or bound to the wild type RNAIII, or to the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9), of hairpin 13 (Δ13) or of hairpin 14 (Δ14). Concentrations of wild type or mutant RNAIII: lane 4, 20 nM; lane 5, 50 nM; lane 6, 100 nM; lane 7, 250 nM. Same legend as in A. (D) DEPC (N7A) modification of unlabeled coa mRNA, free (lane 3) or bound to the wild type RNAIII (lane 4), or the mutant RNAIII deleted of hairpins 7 to 9 (Δ7–9, lane 5), of hairpin 13 (Δ13, lane 6) or of hairpin 14 (Δ14, lane 7) at 200 nM. Lanes U, G, C, A: dideoxy-sequencing reactions performed on coa mRNA. (E) CMCT modification of unlabeled coa mRNA. Same legend as in D. Reactivity changes are indicated by bars on one side of each autoradiography. SD is for Shine-Dalgarno sequence and H7 is for hairpin 7 of RNAIII.