Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression
Figure 1
Effect of mutations in the catalytic site of Staphylococcus aureus RNase III.
(A) Amino acid sequence alignment of RNase III from S. aureus and Escherichia coli. Acidic amino acids colored in green and red are conserved residues present in the catalytic site of E. coli RNase III. The two mutations (D63 to A and E135 to A) generated in S. aureus RNase III are shown in red. (B) RNase III cleavage assays were performed on 5′ end-labeled spa mRNA using the wild-type enzyme (RNase III-wt), the E135A, and the D63A RNase III mutant enzymes. Lanes 1, 2: incubation controls set in the absence of RNase III. Cleavage reactions were performed in the presence of Mg2+ with increasing concentrations of RNase III WT (lanes 3–5), the E135A (lanes 7–9) or D63A (lanes 11–13) mutant proteins: (lanes 3, 7, 11) 0.165 µM; (lanes 4, 8, 12) 0.33 µM; (lanes 5, 9, 13) 0.66 µM. Control reactions included Ca2+ and 0.66 µM of RNase III WT (lane 6), E135A (lane 10) or D63A (lane 14) mutants. Lanes T, L: RNase T1 and alkaline ladders, respectively, under denaturing conditions. Arrows denote the positions of RNase III cleavages which are shown in the secondary structure of the RNase III-binding site on spa mRNA. (C) Binding of the wild-type (RNase III-wt) and mutant (E135A) enzymes to 5′ end-labeled spa mRNA as visualized by gel retardation assays. Lane (−): incubation control of the free RNA in the absence of RNase III. Increasing concentrations of RNase III WT (0.05, 0.1 and 0.2 µM) or RNase III-E135A (0.08, 0.16, 0.32 and 0.49 µM) were added to 5′ end-labeled spa mRNA in a buffer containing Ca2+ instead of Mg2+. (D) RNase III-Flag protein levels at mid-logarithmic phase were monitored in various S. aureus strains: Δrnc mutant strain, Δrnc mutant strain complemented with the Flag-E135A mutant, the Flag-wt RNase III and the Flag-D63A mutant. Cells were collected before or after induction with CdCl2 for 90 min. 40 µg of total protein were resolved on an SDS-PAGE gel. The western blot was performed with an anti-Flag monoclonal antibody.