All NMR spectra were recorded at 283 K in 2 mM MgCl2, 50 mM KCl, 25 mM potassium phosphate (pH 6.2). 1 equivalent of each RNA (GSW10-89 (=GSWPA), S2trans (=Ttrans) and S3trans(=Htrans), respectively) was used, 4 equivalents of ligand were added. The following selective labelling scheme was explored: 15N-G,U GSW10-89, 14N-G,U S2trans and 14N-G,U S3trans. Signals originating from GSW10-89 and S2trans or S3trans were separated using in x-filter 1D experiments (Weixlbaumer et al., 2013) (top). The 1H,15N-HSQC spectra (bottom) report on the interactions in the aptamer domain. (a) In the elongated aptamer domain GSW10-89, the helix PA was formed (U17–G79) and the loop-loop interaction reporter G32 was detected. (b) GSW10-89 and ligand (1:4) ligand binding was monitored by appearance of signals U47, U49 and U51. (c) GSW10-89 and S2trans (1:1): Sequence S2trans caused the PA reporter signals U17(P1) and G79(P1) to decrease, AT formation was followed by appearance of signals of G79(P4), U81(P4) and G82(P4). (d) GSW10-89, S2trans and ligand (1:1:4): Addition of ligand to GSW10-89-S2trans resulted in dissociation of the complex (decreasing signals for G79(P4), U81(P4) and G82(P4) signals) and reformation of the PA helix (U17(P1) and G79(P1) signals). Ligand binding reporters U47, U49 and U51 were detected. However, in presence of 4 equivalents of ligand, AT helix reporter signals were significant. (e) GSW10-89, S2trans and S3trans (1:1:1) Addition of S3trans to GSW10-89-S2trans resulted in complete dissociation of the complex (G79(P4), U81(P4) and G82(P4) signals) and reformation of the PA helix (U17(P1) and G79(P1) signals). In contrast to ligand addition, 1 equivalent of S3trans was sufficient to disrupt the antiterminator mimic. (f) GSW10-89, S2trans, S3trans and ligand (1:1:1:4): Ligand binding to GSW10-89 in presence of the terminator helix P6 (=TH) equals ligand binding to GSW10-89 (GSWPA) alone (b).