Figure 2.

Loc1p induces conformational changes in nucleic acids. (A) Fluorescence intensity of a stem–loop molecular beacon (MB-D16) is not changed upon titration with MBP-Loc1p, indicating that Loc1p is unable to change the secondary structure of this RNA. (B) Fluorescence intensity of a single-stranded molecular beacon (MB-A5) is decreased upon titration of Loc1p, indicating the induction of conformational changes. (C) In the presence of antisense RNA (anti-MB-D16), titration of Loc1p changes the fluorescence intensity of the stem–loop shown in panel (A). This suggests that Loc1p promotes the formation of the energetically favored structure. Left: For steady-state experiments, the molecular beacon was pre-incubated with antisense RNA before Loc1p was added. Right: In time-course experiments, Loc1p, MBP or buffer alone was pre-incubated with the molecular beacon prior to addition of antisense RNA. Addition of 1 μM Loc1p increases the reaction rate ∼7-fold, suggesting that Loc1p promotes the formation of the energetically most favored state. (D) Comparative TAR annealing assay showing that MBP-Loc1p and Imp4p promote annealing of dsDNA while Hfq displaces dsTAR DNA under these experimental conditions. Radioactively labeled TAR(+) DNA was incubated with TAR(−) DNA at 37°C and with MBP-Loc1p, SUMO-Hfq, SUMO-Imp4p or buffer (negative control, 37°C). As a positive control, the sample was incubated at 70°C for heat-induced annealing. While 5 nM of MBP-Loc1p is sufficient for complete annealing, Imp4p requires >3 μM to reach a comparable activity. All experiments were performed independently at least three times.