Figure 4.

Direct interaction between RydC and the csgD mRNA; ternary complex formation between RydC Hfq and its mRNA target. (A) Schematic representation of the csgD mRNA 5′-domain emphasizing three RNA constructs. The csgD mRNA₂₁₅ corresponds to the 215 nt from the 5′-end of the mRNA (red), emphasizing the SD and AUG translation initiation signals. In the csgD mRNA₁₀₀ variant (blue), 115 nt from the csgD mRNA 5′-end were deleted. The 5′-UTR of the csgD mRNA (the sequence between the black brackets) was deleted in mutant csgD mRNA_Δ5′-UTR, therefore starting at G₊₃ (green). (B) Complex formation between RydC and each of the three csgD mRNA constructs. Native gel retardation assays of purified labelled RydC with increasing amounts of purified unlabelled csgD mRNA₂₁₅, csgD mRNA₁₀₀ or csgD mRNA_Δ5′-UTR are shown. The csgD mRNA construct/RydC molar ratios are indicated below each lane. Competition assays were performed with a 100-fold molar excess of unrelated purified SprD RNA (18) in the presence of each of the csgD mRNA₂₁₅ and csgD mRNA₁₀₀ constructs. (C) Ternary complex formation between RydC, csgD mRNA₂₁₅ and Hfq. Left panel: Native gel retardation assays show complex formation between labelled RydC and increasing amounts of unlabelled csgD mRNA₂₁₅ (at a 1- to 50-fold excess as compared with RydC) in the presence or absence of purified Hfq. Hfq is at a 1:1 molar ratio with RydC. The asterisks indicate the ‘RydC*/csgD mRNA’ molar ratio used to perform the competition assays with a 10- to 100-fold molar excess of unlabelled RydC. Right panel: csgD mRNA₂₁₅ interacts with Hfq in the absence of RydC in vitro. Hfq is at a 20:1 molar ratio with the mRNA. The csgD mRNA adopts two conformations on a native gel.