Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

Abstract

S-adenosylmethionine (SAM) riboswitches are widespread in bacteria, and up to five different SAM riboswitch families have been reported, highlighting the relevance of SAM regulation. On the basis of crystallographic and biochemical data, it has been postulated, but never demonstrated, that ligand recognition by SAM riboswitches involves key conformational changes in the RNA architecture. We show here that the aptamer follows a two-step hierarchical folding selectively induced by metal ions and ligand binding, each of them leading to the formation of one of the two helical stacks observed in the crystal structure. Moreover, we find that the anti-antiterminator P1 stem is rotated along its helical axis upon ligand binding, a mechanistic feature that could be common to other riboswitches. We also show that the nonconserved P4 helical domain is used as an auxiliary element to enhance the ligand-binding affinity. This work provides the first comprehensive characterization, to our knowledge, of a ligand-controlled riboswitch folding pathway.

Folding of the SAM-I riboswitch

“…For example, Mg 2+ binding to the aptamer promotes the formation of the pseudoknot interaction, which was previously shown to be also stabilized in presence of SAM. 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). 23 In agreement with this model, it was previously found that the folding of the kink-turn motif located in the P2 helical domain is crucial for the structure and ligand binding activity of the SAM-I aptamer. 46,50 Our study also revealed that SAM binding to the aptamer produces a second folding transition involving the stacking of stems P1 and P4.…”

“…46,50 Our study also revealed that SAM binding to the aptamer produces a second folding transition involving the stacking of stems P1 and P4. 23 SAM binding was also previously shown to reorganize regions J1/2 and J3/4, 33,54 consistent with the formation of the native RNA-ligand complex as observed in the crystal structure. 49 …”

“…23 In this study, it was found that the SAM-I aptamer follows a twostep hierarchical folding that is selectively…”

“…However, upon SAM binding, the riboswitch folds into an alternative secondary structure allowing the formation of the P1 stem, which in turn allows formation of a terminator structure (t) to attenuate transcription. Adapted from Heppell et al 23 only strongly increased upon addition of SAM. This observation suggests that SAM binding stabilizes the aptamer in the native state, termed F NS (Fig.…”