RNA Tertiary Interactions in a Riboswitch Stabilize the Structure of a Kink Turn

Summary The kink turn is a widespread RNA motif that introduces an acute kink into the axis of duplex RNA, typically comprising a bulge followed by a G⋅A and A⋅G pairs. The kinked conformation is stabilized by metal ions, or the binding of proteins including L7Ae. We now demonstrate a third mechanism for the stabilization of k-turn structure, involving tertiary interactions within a larger RNA structure. The SAM-I riboswitch contains an essential standard k-turn sequence that kinks a helix so that its terminal loop can make a long-range interaction. We find that some sequence variations in the k-turn within the riboswitch do not prevent SAM binding, despite preventing the folding of the k-turn in isolation. Furthermore, two crystal structures show that the sequence-variant k-turns are conventionally folded within the riboswitch. This study shows that the folded structure of the k-turn can be stabilized by tertiary interactions within a larger RNA structure.


Figure S2, related to Figure 3. Isothermal titration analysis of SAM binding to the natural sequence riboswitch and further variants with k-turn substitutions.
A solution of SAM was titrated into a SAM-I riboswitch RNA solution, and the heat evolved was measured by ITC as the power required to maintain zero temperature difference with a reference cell. Integration over time gives the heat required to maintain thermal equilibrium between cells. In each case the upper panel shows the raw data for sequential injections of 8 µL volumes (following an initial injection of 1 µL) of a 100 µM solution of SAM into a 1.4 mL 10 µM RNA solution in 50 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl 2 . This represents the differential of the total heat (ie enthalpy ∆H° under conditions of constant pressure) for each SAM concentration. The lower panels present the integrated heat data fitted (where possible) to a single-site binding model. The thermodynamic parameters calculated are summarized in Table S2. The ITC analysis was performed for the SAM-I riboswitch in which the k-turn sequence was modified as follows: A. Unmodified k-turn.
B. The A1nC, G2nU, G3nU triple substitution. These changes convert the k-turn into a simple threenucleotide bulge.  Table S1. Summary of the folding properties of the SAM-I k-turn and sequence variants analyzed as duplex species by FRET as a function of Mg2+ concentration (see Figure 2). concentration. This can therefore be regarded as essentially not folded.      Values of FRET efficiency (E FRET ) were measured using the acceptor normalization method (Clegg, 1992) implemented in MATLAB. E FRET as a function of metal ion concentration was analyzed on the basis of a model in which the fraction of folded molecules corresponds to a simple two-state model for ion-induced folding, ie where E 0 is the FRET efficiency of the RNA in the absence of added metal ions, ∆E FRET is the increase in FRET efficiency at saturating metal ion concentration, [M] is the prevailing Mg 2+ ion concentration, K a is the apparent association constant for metal ion binding and n is a Hill coefficient. Data were fitted to this equation by nonlinear regression. The metal ion concentration at which the transition is half complete is given The sequences used in the FRET analyses were : bulged strand: Fluorescein-CCAGUCAGUCCCGACGAAACCUGUCAGG non-bulged strand: Cy3-CCUGACAGGUGGAGGGACUGACUGG Nucleotide substitutions were introduced as required.

Isothermal titration calorimetry
Microcalorimetric measurements of SAM binding to the SAM-I riboswitch and variants were performed by isothermal titration calorimetry (ITC) using a MicroCal VP-ITC (GE Healthcare) at 30°C as described by Montange et al (Montange et al., 2010). where ΔH is the change in enthalpy, v is the reaction volume, K a is the association constant for SAM binding, and [SAM] i is the SAM concentration at the i th injection Each titration was performed three times and ∆H° and ∆S° values were averaged. Free energy (∆G°) and the dissociation constant for SAM binding (K d ) were calculated from : ∆H° -T∆S° = ∆G° = -RTlnK d

[3]
where R is the gas constant and T is the temperature (K).

X-ray crystallography
The SAM-I riboswitch variants were crystallized using the hanging drop method. Crystal plates were set up by mixing 1 µL of mother liquor with 1 µL of 400 µM RNA plus 1 mM SAM (Sigma Aldrich) in 40 mM Na-cacodylate (pH 7.0). Drops were seeded using the micro seeding technique with a seed stock obtained from crystal plates containing the unmodified RNA. The mother liquor of the drop that yielded the crystal of the G2nU, were collected on ID14-4 (G2nU, G3nU) and BM-14 (G2nA) at the European Synchrotron Radiation Facility in Grenoble, France. Data were indexed, integrated and scaled using HKL2000 (Otwinowski et al., 2003) for G2nU, G3nU and MOSFLM/Scala for the G2nA structure from the CCP4 suite of programs (CCP4, 1994). Both structures were solved by performing a molecular replacement using with the RNA plus SAMligand structure PDB entry 3gx5 (Montange et al., 2010) as a preliminary model. The structures were refined using REFMAC5 from the CCP4 suite of programs, and the model was built using COOT (Emsley and Cowtan, 2004). The composite omit map was calculated using Phenix (Adams et al, 2010)