Base Pairing Interaction between 5′- and 3′-UTRs Controls icaR mRNA Translation in Staphylococcus aureus
Figure 3
icaR 3′-UTR post-transcriptionally modulates IcaR expression.
(A) Schematic representation of chromosomal 3′-UTR deletion. Note that the transcriptional terminator is not affected by the deletion. (B) qRT-PCR analysis of icaR mRNA levels in S. aureus 15981 wild type and Δ3′-UTR strains grown in TSB-gluc at 37°C until exponential phase (OD600 nm = 0.8). The gyrB transcript was used as an endogenous control, and the results were expressed as the n-fold difference relative to the control gene (2−ΔCt, where ΔCt represents the difference in threshold cycle between the target and control genes). (C) A representative Northern blot showing icaR mRNA of wild type and Δ3′UTR strains grown in TSB-gluc at 37°C until exponential phase (OD600 nm = 0.8). Lower panel shows 16S ribosome band stained with ethidium bromide as loading control. (D) A representative Western blot showing IcaR protein levels expressed from strains shown in panel A. The 3XFLAG tagged IcaR protein was detected with commercial anti-3XFLAG antibodies. Numbers below the image show relative band quantification according to densitometry analysis performed with ImageJ (http://rsbweb.nih.gov/ij/). A Coomassie stained gel portion is shown as loading control. (E) Schematic representation of plasmid constructions constitutively expressing the 3XFLAG tagged IcaR protein from the whole icaR mRNA or the mRNA carrying the 3′-UTR deletion. (F) A representative Western blot showing IcaR protein levels of strains shown in panel E. The 3XFLAG tagged IcaR protein was detected with commercial anti-3XFLAG antibodies. Densitometry analysis is also shown. On the left, a Coomassie stained gel portion is shown as loading control.