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.

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

“…This analysis was enabled by the inclusion of the leader, which allows the riboswitch to behave well at higher RNA concentrations. We confirmed that wildtype (WT) VC1-2 containing the leader sequence binds glycine noncooperatively with low micromolar affinity, in agreement with other recent reports (Kladwang et al 2012;Sherman et al 2012;Baird and Ferré-D'Amaré 2013). Because the inclusion of the leader sequence dramatically changes the ligand-binding activity of the riboswitch, we wished to verify if each ligand-binding site in the tandem riboswitch with leader binds to a separate molecule of glycine.…”

“…At RNA concentrations ∼30 times the K d , the WT tandem riboswitch containing the leader sequence binds 1.8 equivalents of glycine (Table 1), in agreement with the original studies on the glycine riboswitch, but in contrast to the recent report of a single equivalent bound as reported using isothermal titration calorimetry (ITC) (Baird and Ferré-D'Amaré 2013). The disparity might arise from a variety of differences between the techniques, including longer equilibration times or refolding the riboswitch in the presence of the ligand.…”

“…1A; Mandal et al 2004). The two aptamers each bind glycine with micromolar dissociation constants (1-30 µM K d ), and, like many other riboswitches, the glycine riboswitch undergoes conformational compaction upon the addition of glycine Lipfert et al 2007; Kwon and Strobel 2008;Baird and Ferré-D'Amaré 2013;Zhang et al 2014). It is the only known riboswitch where two aptamer domains control a single expression platform Breaker 2011).…”

“…1A), the inclusion of which eliminates cooperativity between the aptamer domains, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch (Kladwang et al 2012;Sherman et al 2012). A recent calorimetry study, while showing that the leader helix promotes ligand binding and riboswitch compaction, has further questioned whether the tandem riboswitch with the leader P0 helix binds one or two equivalents of glycine (Baird and Ferré-D'Amaré 2013).…”

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…When in water only, RNA is expected to be unstructured, and W-band spectrum ( Figure 4C top and S8) with a SNR value of 18:1 reveal disorder, with spectral simulation giving a correlation time of 0.4 ns. It has previously been shown that, upon addition of salts and ligands, the VC glycine riboswitch folds with formation of the kink turn [53,54,63,64] where, based upon comparisons to X-ray models of other k-turn motifs [65,66], the modified U2-base is expected to become base paired/base stacked. Hence, a decrease in dynamics (an increase in correlation time) is anticipated.…”