Modulation of quaternary structure and enhancement of ligand binding by the K-turn of tandem glycine riboswitches

Abstract

Most known glycine riboswitches have two homologous aptamer domains arranged in tandem and separated by a short linker. The two aptamers associate through reciprocal "quaternary" interactions that have been proposed to result in cooperative glycine binding. Recently, the interaptamer linker was found to form helix P0 with a previously unrecognized segment 5' to the first aptamer domain. P0 was shown to increase glycine affinity, abolish cooperativity, and conform to the K-turn motif consensus. We examine the global thermodynamic and structural role of P0 using isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS), respectively. To evaluate the generality of P0 function, we prepared glycine riboswitch constructs lacking and including P0 from Bacillus subtilis, Fusobacterium nucleatum, and Vibrio cholerae. We find that P0 indeed folds into a K-turn, supports partial pre-folding of all three glycine-free RNAs, and is required for ITC observation of glycine binding under physiologic Mg(2+) concentrations. Except for the unusually small riboswitch from F. nucleatum, the K-turn is needed for maximally compacting the glycine-bound states of the RNAs. Formation of a ribonucleoprotein complex between the B. subtilis or the F. nucleatum RNA constructs and the bacterial K-turn binding protein YbxF promotes additional folding of the free riboswitch, and enhances glycine binding. Consistent with the previously reported loss of cooperativity, P0-containing B. subtilis and V. cholerae tandem aptamers bound no more than one glycine molecule per riboswitch. Our results indicate that the P0 K-turn helps organize the quaternary structure of tandem glycine riboswitches, thereby facilitating ligand binding under physiologic conditions.

FIGURE 1.

The P0 helix of glycine riboswitches is a K-turn. (A) Tandem glycine riboswitch aptamers joined by a single-stranded linker, denoted I–II (left), and by the recently discovered (Kladwang et al. 2012; Sherman et al. 2012) K-turn, denoted I–II^Kt (right). The sequence of the K-turn is given for each of the three I–II^Kt examined. (B) EMSA analysis of three I–II^Kt constructs using the L7Ae and YbxF K-turn binding proteins.

FIGURE 2.

ITC analyses of RNA folding and glycine binding. (A) Mg²⁺ titration of the three I–II^Kt RNAs used in this study. For clarity, data for FnI–II^Kt and VcI–II^Kt are offset by 0.4 kcal/mol and 0.8 kcal/mol, respectively, in this panel only. (B) ITC titrations of glycine into VcI–II (left) and VcI–II^Kt (right) in 5 mM Mg²⁺. (C) ITC titrations of glycine into BsI–II (left) and BsI–II^Kt (right) in 5 mM Mg²⁺, including a titration in which the final molar ratio (glycine:RNA) in the solution is >8 (inset).

FIGURE 3.

SAXS analyses of VcI–II and VcI–II^Kt. (A) Comparison of R_g in the absence and presence of saturating glycine in 5 mM and 10 mM Mg²⁺. (B) Kratky and (C) P(r) plots for experiments performed in 5 mM Mg²⁺ with VcI–II and VcI–II^Kt in the absence and presence of glycine.

FIGURE 4.

SAXS analyses of B. subtilis tandem glycine aptamers. (A) R_g in the absence and presence of saturating glycine in 1.5 mM, 5 mM, and 10 mM Mg²⁺ for BsI–II, BsI–II^Kt, and BsI–II^Kt + YbxF. Sample marked with asterisk exhibited partial oligomerization (Materials and Methods). (B) Kratky and (C) P(r) plots for experiments performed in 5 mM Mg²⁺ with BsI–II and BsI–II^Kt in the absence and presence of glycine. (D) Kratky plots (in 5 mM Mg²⁺, except where noted) demonstrating the effect of Mg²⁺ and YbxF on the structure of BsI–II^Kt in the absence of glycine. VcI–II^Kt plot is reproduced from Figure 3D for additional comparison. (E) Kratky plots demonstrating the effect of glycine in 1.5 mM and 5 mM Mg²⁺.

FIGURE 5.

ITC analyses of FnI–II and FnI–II^Kt. (Left) Titration of glycine into FnI–II. A small heat signal is observed upon injection of glycine (inset) and is very slow to re-equilibrate (>10 min). (Right) Glycine binding by FnI–II^Kt results in a binding isotherm.

FIGURE 6.

SAXS analyses of FnI–II, FnI–II^Kt, and FnI-II^Kt + YbxF. (A) R_g in the absence and presence of saturating glycine in 5 mM and 10 mM Mg²⁺. (B) Kratky and (C) P(r) plots from data collected at 5 mM Mg²⁺ demonstrate glycine-binding-induced compaction. (D) Kratky plots comparing the effect of Mg²⁺, YbxF, and glycine on folding of FnI–II^Kt (in 5 mM Mg²⁺, except where noted). (E) Comparison of the experimental X-ray scattering profile for FnI–II^Kt with the scattering profile calculated (Materials and Methods) from the crystal structure (Butler et al. 2011) of FnI–II.