Multiple conformations of SAM-II riboswitch detected with SAXS and NMR spectroscopy

Abstract

Riboswitches are a newly discovered large family of structured functional RNA elements that specifically bind small molecule targets out of a myriad of cellular metabolites to modulate gene expression. Structural studies of ligand-bound riboswitches by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy have provided insights into detailed RNA–ligand recognition and interactions. However, the structures of ligand-free riboswitches remain poorly characterized. In this study, we have used a variety of biochemical, biophysical and computational techniques including small-angle X-ray scattering and NMR spectroscopy to characterize the ligand-free and ligand-bound forms of SAM-II riboswitch. Our data demonstrate that the RNA adopts multiple conformations along its folding pathway and suggest that the RNA undergoes marked conformational changes upon Mg2+ compaction and S-adenosylmethionine (SAM) metabolite binding. Further studies indicated that Mg2+ ion is not essential for the ligand binding but can stabilize the complex by facilitating loop/stem interactions. In the presence of millimolar concentration of Mg2+ ion, the RNA samples a more compact conformation. This conformation is near to, but distinct from, the native fold and competent to bind the metabolite. We conclude that the formation of various secondary and tertiary structural elements, including a pseudoknot, occur to sequester the putative Shine–Dalgarno sequence of the RNA only after metabolite binding.

Common themes and differences in SAM recognition among SAM riboswitches

“…These experiments showed that even in the absence of SAM, a stem-loop structure samples a pseudoknot (bound-like) state by making tertiary interactions between the 3′ strand and the loop. This state sampling is further supported by recent SAXS and NMR data[43]. Though the distribution of states depends on the concentration of Mg 2+ , the populations were roughly equal in the smFRET studies performed with 2 mM Mg 2+ in the absence of SAM[44].…”

“…SAM-II is stabilized in a near bound state by Mg 2+ , but like SAM-I, it only reaches its final stabilized form in the presence of SAM[43, 44]. SAM-III is also a dynamic structure that undergoes large conformational sampling between three discrete states: translation “on”, “ready”, and “off” states.…”

A magnesium-induced triplex pre-organizes the SAM-II riboswitch

“…Here RNA elutes after longer retention times with increasing Mg 2+ concentration ([Mg 2+ ]) in the mobile phase [40]. Bigger elution volume signifies decreasing hydrodynamic radius of a monomeric RNA molecule.…”

“…A total of 1024x16 complex points were recorded [40] with 32 transients with a recovery delay of 1.5 s for a total experimental time of approximately 12 hr for each spin-lock field. A CEST saturation period of 100 ms was used for the base and 200 ms for ribose.…”

Characterizing excited conformational states of RNA by NMR spectroscopy

“…Although Tier-0 excited states can often be directly observed by NMR, as in the previous two examples, some Tier-0 excited states do not give observable NMR signals and their existence is often inferred from various biophysical and biochemical measurements [49,50]. These states are ‘invisible’ to NMR, which can be due to either a low population and/or a short lifetime.…”