Folding of the Thiamine Pyrophosphate (TPP) Riboswitch

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TPP riboswitches regulate the expression of thiamine-synthesis (thi) genes through a variety of mechanisms, all of which involve binding of TPP to a structured aptamer formed in the untranslated region (UTR) of a thi mRNA. We used a high-resolution, single-molecule optical trapping assay to characterize mechanically the folding of the TPP riboswitch aptamer located in the 3’UTR of the thiC gene of Arabidopsis thaliana. Each RNA molecule, containing either the complete aptamer sequence or a portion thereof, was transcribed in vitro, annealed to DNA handles via single-stranded overhangs, and placed in a “dumbbell” experimental geometry1. By applying tension to the ends of the RNA molecule under equilibrium conditions and measuring the corresponding extensions, we observed transitions among several well-defined folding states, which we discuss in the context of secondary and tertiary structures formed by the aptamer2. One low-force state of the full aptamer, corresponding to the formation of structural elements located near the three-helix junction, was abolished by mutating a single nucleotide believed to participate in specific tertiary contacts within the junction2,3. We observed that the mutant aptamer does not bind TPP or other substrates (thiamine, thiamine monophosphate), and that the wild-type aptamer only binds substrates concomitant with entry into the fully-folded state. We also studied the energetics of substrate binding under non-equilibrium conditions by rapidly increasing or decreasing the extension of the aptamer and measuring the hysteresis in force. The number of phosphates on the substrate modulated the amount of work required to induce substrate unbinding, the height and location of the energy barrier to substrate unbinding, and the amount of RNA released.1. Greenleaf WJ, et al (2005). PRL 95, 208102.2. Thore S, et al (2006). Science 312, 1208-1211.3. Sudarsan N, et al (2005). Chemistry & Biology 12, 1325-1335.