Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression
Figure 4
RNase III processes the 5′ untranslated region of cspA mRNA.
(A) IGB representation of the cspA locus. Same legend as in Figure 2A is applied. The +1 site identified by primer extension in the Δrnc strain is indicated by a red arrow (U-113). (B) Expression of cspA mRNA forms after 4 h of growth at 37°C in strains RN6390 (WT), Δrnc and Δrnc expressing the E135A, WT or D63A Flag-tagged RNase III. CspAL corresponds to the longest form of the mRNA while cspAS corresponds to the processed form of the mRNA. (C) Growth curves of RN6390 (WT) and Δrnc mutant strains (Δrnc). Two cultures were grown at 37°C (black diamonds: RN6390; black triangles: Δrnc) while two other cultures (black squares: RN6390; black circles: Δrnc) were transferred to 15°C (t0) after 120 min of growth at 37°C, then incubated for an additional 120 min at 15°C, and retransferred to 37°C at time t3. In this experiment, a mild growth defect was observed for the Δrnc strain. However, this defect was not reproducible and could be attributed to a higher level of cell aggregation in this strain during the late exponential phase of growth. Northern blot analyses showing the expression of cspA mRNA at 37°C or at 15°C at the indicated time-points are depicted in the insets. Lanes 1, 2: RNA samples prepared from RN6390 or Δrnc mutant strains, respectively. Lanes t-1 to t5: incubation times of cell cultures as shown in the growth curves. A DIG-labeled DNA probe (amplified using the oligonucleotides 286 and 16) was used to detect cspA mRNA and the autoradiography was revealed after several seconds. (D) Primer extension analysis performed on total RNAs isolated from cells grown at 37°C for 3 h and 4 h. Lane 1: RN6390 strain; lane 2: Δrnc strain. The 5′ end detected in each strain is indicated. The nucleotides are numbered relatively to the AUG start codon. Lanes C, U, A, G: sequencing ladders. Primer extension was carried out using the 5′ end-labeled oligonucleotide 378 (Table S8). (E) RNase III cleavage of unlabeled cspAL mRNA. The reactions were done in the absence (−) and in the presence of RNase III (+, 0.33 µM; ++ 0.65 µM) in a buffer containing Mg2+ or Ca2+. Lanes C, U, G, A: sequencing reactions. The same oligonucleotide 378 was used for reverse transcription to analyze the cleavage sites. The RNase III cut at position A-88 appears as a faint band because the enzymatic reaction is too strong. Arrows denote the specific RNase III-induced cleavages at U-53 and A-88 (relatively to the AUG). Lane (−): incubation control in the absence of RNase III. The cleavages were assigned after primer extension using the 5′ end-labeled oligonucleotide 16. (F) Secondary structure of cspAL is deduced from structure probing experiments. The structure of the 5′UTR of cspAS is shown in the inset. The grey arrow corresponds to the RNase III cleavage sites obtained in vitro while the red arrow represents the reverse transcriptase stop, which was assigned by primer extension in RN6390 (WT). The Shine and Dalgarno (SD) sequence and the AUG strat codon are indicated in red.