Base Pairing Interaction between 5′- and 3′-UTRs Controls icaR mRNA Translation in Staphylococcus aureus
(A) Half-life measurement of icaR wild type and Δ3′-UTR mRNAs constitutively expressed in the wild type and Δrnc mutant strains. These strains were grown in TSB-gluc at 37°C until exponential phase (OD600 nm = 0.8) and then rifampicin (300 µg/ml) was added. Samples for RNA extraction were taken at the indicated time points (min). The experiment was repeated three times and representative images are shown. (B) Levels of icaR mRNA were quantified by densitometry of Northern blot autoradiographies using ImageJ (http://rsbweb.nih.gov/ij/). Each of mRNA levels was relativized to mRNA levels at time 0. The logarithm values of relative mRNA levels were subjected to linear regression analysis and plotted as a function of time. Error bars indicate the standard deviation of mRNA levels from three independent experiments. Dashed lines indicate the time at which 50% of mRNA remained. The half-life of mRNAs is shown above of X-axis. (C) Representative Western blot showing IcaR protein levels in different mutant strains constitutively expressing the 3XFLAG tagged IcaR from the PblaZ promoter. Tagged IcaR protein was detected with commercial anti-3XFLAG antibodies. On the left, a Coomassie stained gel portion is shown as loading control. rnc, double-stranded endoribonuclease RNase III; pnp, polynucleotide phosphorylase PNPase; yqfR, (SAOUHSC_01659), ATP-dependent RNA helicase containing a DEAD box domain; hfq, RNA chaperone, host factor-1 protein.