Base Pairing Interaction between 5′- and 3′-UTRs Controls icaR mRNA Translation in Staphylococcus aureus
Figure 5
A UCCCCUG motif is necessary for the interaction between the 3′-UTR and the Shine-Dalgarno region of icaR mRNA in vitro.
(A) Schematic representation of the 5′-3′-UTRs interaction. A UCCCCUG motif located at the 3′-UTR pairs the UAGGGGG Shine-Dalgarno region located at the 5′-UTR. The numbers indicate the relative position of the nucleotides in the full-length icaR mRNA (B) Substitution of the 894UCCCCUG900 motif by 894AGGGGAC900, disrupts the pairing predicted by Mfold program. (C) Introduction of a compensatory mutation sequence (57GUCCCCU63) in the 5′-UTR, complementary to the substituted 894AGGGGAC900 motif, restores complex formation. (D) Gel shift analysis of the 5′ and 3′-UTR icaR mRNA interaction. The 32P-labeled 5′-UTR fragment (1–117-nt) was incubated with increasing concentrations of unlabeled 3′-UTR (3′-UTR WT) or substituted 3′-UTR (3′-UTR-AGGGGAC) (838–957-nt). (E) Similarly, the 32P-labeled compensatory-5′-UTR fragment (32P-5′-UTR-GUCCCCU) was incubated with increasing concentrations of unlabeled 3′-UTR (3′-UTR WT) or substituted 3′-UTR (3′-UTR-AGGGGAC). After 30 min of incubation at 37°C, the mixture was analysed by electrophoresis in a native 5% polyacrylamide gel and PhosphorImager (Molecular Dynamics).