Figure 1.
RNAIII-dependant regulation of coa mRNA in vivo.
β-galactosidase activity detected from different gene fusions. β-galactosidase activity measured from PrpoB-coa (+1/+126)::lacZ fusions in various S. aureus strains: LUG1467 (rnaIII+, wt), LUG1446-Δrnc (deletion of rnc gene encoding RNase III), LUG1445-Δhfq (deletion of hfq gene), LUG1457 (ΔrnaIII), and LUG1457 transformed with the plasmid expressing the wild type RNAIII, the 3′ domain, RNAIII-Δ13 (RNAIII deleted of hairpin 13), or RNAIII-Δ7–9 (RNAIII deleted of hairpins 7 to 9), or with the plasmid containing no insert (pE194). The β-galactosidase activity was normalized for total cell density and is represented as a percentage of the uninhibited control (LUG1457). The results represented a mean of three independent experiments.
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.
Figure 3.
Structure of the RNAIII-coa mRNA complex.
(A) Summary of the enzymatic cleavages and chemical reactivities of nucleotides of coa mRNA. Enzymatic cleavages are given as follows: RNase T1 (black arrow), and RNase V1 (white arrowhead) moderate, (black arrowhead) strong cleavage. Chemical modifications of cytosines at N3, and adenines at N1 by DMS, of uridines at N3 and guanines at N1 by CMCT, and of adenines at N7 by DEPC: full and dashed circled nucleotides are for strong and moderate reactivity, respectively. No symbol is for non reactive, nd is for not determined due to non-specific cleavages or pauses of RT in the incubation control. The reactivity of A at N7 is reported on the secondary structure shown in the insert. Reactivity changes induced by the binding of RNAIII are indicated as follows: black circles denote strong protection, enhancements and new RNase V1 cleavages are represented by asterisks. (B) Secondary structure model of the RNAIII-coa mRNA complex showing the reactivity changes induced by complex formation: RNase V1 (white and black arrowheads), RNase III cleavages (double arrow). Effect of RNAIII binding: Protected nucleotides are squared in black and the nucleotides, which become accessible, are squared in red. (C) The topology of the loop-loop interaction built by graphic modeling based on the probing data. The hairpin III of coa mRNA is in green and the hairpin 7 of RNAIII in red. The RNase III cleavages are shown in blue.
Figure 4.
RNAIII binds efficiently to coa mRNA in vitro.
(A) Determination of the apparent dissociation constant for RNAIII-coa mRNA complex. 5′-end-labeled coa mRNA was incubated alone (−) or with various concentrations of unlabeled wild type RNAIII, the 3′ domain, RNAIII-Δ7-9, and RNAIII-Δ13 (1, 5, 10, 20, 50, 100, 200, and 250 nM). The fraction of labeled coa mRNA associated with RNAIII or its derivatives was calculated from the counts in the corresponding band relative to the total counts in the lane. The Kd value was estimated as the concentration of RNAIII allowing 50% of coa mRNA binding. (B) Binding rate constant for various RNAIII-coa mRNA complexes as determined from three independent experiments. 5′-end-labeled coa mRNA (0.1 nM) was incubated with unlabeled RNAIII (20 nM), RNAIII-Δ7-9 (20 nM), and RNAIII-Δ13 (20 nM) at 37°C. Aliquots were withdrawn at various times (from 0 to 350 sec). The percentage of free coa mRNA was plotted as a function of time to estimate the association rate constant according to [40]. The values for the binding rate constants are the means of three independent experiments: 1.1×105 M−1 s−1 (RNAIII), 9.5×105 M−1 s−1 (RNAIII-Δ7-9), and 1.1×104 M−1 s−1 (RNAIII-Δ13).
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.
Figure 6.
Schematic view of RNAIII-mediated repression of coa mRNA.
RNAIII binds to its target mRNA masking the RBS and part of the coding sequence. Binding of RNAIII hinders ribosome binding and promotes access to RNase III. SD (Shine-Dalgarno) and AUG are in green. RNAIII is in red, the mRNA target is in black, 30S is for small ribosomal subunit.
Table 1.
Strains and plasmids.