Base Pairing Interaction between 5′- and 3′-UTRs Controls icaR mRNA Translation in Staphylococcus aureus
(A, B) Formation of the ternary complex between icaR 5′-UTR fragment (5 nM), S. aureus 30S ribosomal subunit, and initiator tRNA was monitored in the absence or in the presence of increasing concentrations of wild-type 3′-UTR fragment and substituted 3′-UTR fragment. The toeprint at position +16 is indicated. The quantification of the toeprint (B) was first normalized according to the full-length extension product bands using the SAFA software , and the toeprint signal (given in %) represents the yield of the toeprint obtained in the presence of the competitor RNA versus the yield of the toeprint obtained in the absence of the competitor RNA. (C, D) Formation of the ternary complex with the 5′-UTR fragment and the full-length icaR mRNA molecule was monitored using different S. aureus 30S concentrations. A reverse transcriptase pause at the Shine-Dalgarno (SD) sequence occurring in the full-length icaR mRNA molecule is indicated with an arrow. The quantification of toeprint experiment (D) is described above in B. (E) Schematic representation of plasmid constructions constitutively expressing the 3XFLAG tagged IcaR protein from the different icaR mRNA alleles. (F) A representative Western blot showing IcaR protein levels in strains shown in panel E. The 3XFLAG tagged IcaR protein was detected with commercial anti-3XFLAG antibodies. Band quantification according to densitometry analysis is shown. A Coomassie stained gel portion is shown as loading control.