Structural analysis of the ‘SprD-sbi mRNA’ duplex indicates that SprD binds to the sbi mRNA ribosome binding site.
(A) Secondary structures of the SprD RNA and of the sbi mRNA 5′-end (nts 1–62) from S. aureus N315 based on structural probes in solution that supports each of the proposed structures. Triangles are V1 cuts; arrows capped by a circle are S1 cuts; uncapped arrows are lead cuts. Intensities of cuts and cleavages are proportional to the darkness of the symbols. Structural domains are indicated. The AUG and putative SD sequence are squared on the sbi 5′-end mRNA structure. On the secondary structure models of two isolated RNAs, the nucleotides involved in the structural changes induced by the formation of the ‘SprD-sbi mRNA’ duplex have been circled. (B) Conformational changes of SprD induced by complex formation with the sbi mRNA detected by structural probes. Autoradiograms of cleavage products of 5′-labeled SprD by RNases S1 and V1 in the presence (+) or absence (−) of sbi mRNA. Lanes C, incubation controls; lanes GL, RNase T1 hydrolysis ladder; lanes AL, RNase U2 hydrolysis ladder. The RNA sequence is indexed on the right side. (C) Conformational changes of the sbi mRNA 5′-end induced by complex formation with SprD monitored by structural probes. Indications are as for panel A. (D) Pairing interactions between SprD and the sbi mRNA 5′-end, based on (i) computer prediction, (ii) native gel retardation assays and mutational analyses, (iii) structural mapping of the conformation of SprD in complex with the sbi mRNA 5′-end and (iv) structural mapping of the conformation of the sbi mRNA 5′-end in complex with SprD. Only the structural information concerning the conformation of the duplex is indicated, using similar signs as for panel A. The plus (+) and minus (−) signs indicate respectively the appearance or the disappearance of cleavages by the structural probes when the two RNAs are in duplex.