Free State Conformational Sampling of the SAM-I Riboswitch Aptamer Domain

Abstract

Summary Riboswitches are highly structured elements residing in the 5' untranslated region of messenger RNAs that specifically bind cellular metabolites to alter gene expression. While there are many structures of ligand bound riboswitches that reveal details of bimolecular recognition, their unliganded structures remain poorly characterized. Characterizing the molecular details of the unliganded state is crucial for understanding the riboswitch's mechanism of action because it is this state that actively interrogates the cellular environment and helps direct the regulatory outcome. To develop a detailed description of the ligand free form of an S-adenosylmethionine binding riboswitch at the local and global levels, we have employed a series of biochemical, biophysical, and computational methods. Our data reveals that the ligand binding domain adopts an ensemble of states that minimizes the energy barrier between the free and bound states to establish an efficient decision making brachpoint in the regulatory process.

Folding of the SAM-I riboswitch

“…48 In addition, Mg 2+ binding induces the P2-P3 helical stacking as well as a close juxtaposition between helices P1 and P3, 23 the latter being consistent with SAXS experiments. 27,53 Disruption of the pseudoknot interaction was found to perturb P1-P3 close juxtaposition, which supports the idea that the pseudoknot may be used as a lever to correctly position P3 close to P1, via the P2-P3 helical stacking (Fig. 3A).…”

“…3A), consistent with recent findings obtained by the Batey group. 27 In particular, we have found that the binding of each cofactor is important for the formation of one of the two helical stacks observed in the crystal structure.…”

“…17,24 Intriguingly, some crystal structures of ligand-free aptamers are highly similar to those obtained in their bound form, suggesting that free aptamers may sample various conformations resembling those adopted in presence of the ligand. [25][26][27] Because riboswitches have been identified as promising targets for antibacterial drug development, 28,29 it is imperative to get more information about how riboswitch folding processes are used for recognition of cellular metabolites, and how this is used to modulate gene expression.…”

“…2B). 27,38,49,50 The binding site is constituted by the minor grooves of helices P1 and P3, in which the SAM adenine moiety interacts with a bulge located in the P3 stem. The methionine group of the ligand is involved in interactions with the J1/2 region.…”

“…27,[51][52][53] In particular, using small X-ray scattering (SAXS), the Batey and Ferré-D'Amaré groups have observed that the RNA experiences compactions both in presence of magnesium ions and SAM. 27,53 Furthermore, Batey and colleagues concluded that the aptamer transits from "open" to "closed" conformations in which P1 and P3 are apart or in proximity, respectively. Their results are consistent with the idea that the SAM-I riboswitch samples bound-like conformations in the absence of ligand.…”