Global Regulatory Functions of the Staphylococcus aureus Endoribonuclease III in Gene Expression
Figure 6
The ncRNA RsaA is a target of RNase III.
(A) (Left) The half-life of RsaA was measured as a function of time after rifampicin treatment in the RN6390 and Δrnc strains. A strand specific DIG labeled riboprobe was used to monitor RsaA expression. The oligonucleotides 374 and 376 were used for PCR amplification and the RNA was transcribed in vitro with T7 RNA polymerase. (Right) Quantification of mRNA levels in the RN6390 (WT, diamonds) and Δrnc mutant strains (squares) as a function of time after rifampicin treatment. The value of the mRNA half-life was determined by representing a semi-logarithmic plot of the concentration of the mRNA over time. The value corresponding to the percentage (%) of the remaining mRNA was normalized with the control experiment performed with 5S rRNA. The slope of the best-fit line was then determined to calculate the half-life. Three experiments provided reproducible results. (B) RNase III cleavages of cold RsaA in vitro. 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 performed on RsaAL. (C) The secondary structure of RsaA was experimentally determined [43]. The two RNase III cleavages are represented as follows: empty and black arrows denote weak and strong cleavages, respectively. The organization of the genes corresponds to the annotation of the N315 genome. The ncRNA genes are shown in red.