Crystal structure and ligand-induced folding of the SAM/SAH riboswitch

Abstract

While most SAM riboswitches strongly discriminate between SAM and SAH, the SAM/SAH riboswitch responds to both ligands with similar apparent affinities. We have determined crystal structures of the SAM/SAH riboswitch bound to SAH, SAM and other variant ligands at high resolution. The riboswitch forms an H-type pseudoknot structure with coaxial alignment of the stem–loop helix (P1) and the pseudoknot helix (PK). An additional three base pairs form at the non-open end of P1, and the ligand is bound at the interface between the P1 extension and the PK helix. The adenine nucleobase is stacked into the helix and forms a trans Hoogsteen–Watson–Crick base pair with a uridine, thus becoming an integral part of the helical structure. The majority of the specific interactions are formed with the adenosine. The methionine or homocysteine chain lies in the groove making a single hydrogen bond, and there is no discrimination between the sulfonium of SAM or the thioether of SAH. Single-molecule FRET analysis reveals that the riboswitch exists in two distinct conformations, and that addition of SAM or SAH shifts the population into a stable state that likely corresponds to the form observed in the crystal. A model for translational regulation is presented whereby in the absence of ligand the riboswitch is largely unfolded, lacking the PK helix so that translation can be initiated at the ribosome binding site. But the presence of ligand stabilizes the folded conformation that includes the PK helix, so occluding the ribosome binding site and thus preventing the initiation of translation.

Cobalamin riboswitches are broadly sensitive to corrinoid cofactors to enable an efficient gene regulatory strategy

“…Among the various families of SAM riboswitches, highly specific binding of SAM and exclusion of SAH is achieved by RNA structures that discriminate the charged sulfonium ion of SAM from the uncharged sulfoether of SAH (Price et al, 2014). In contrast, the SAM/SAH riboswitch class attains effector promiscuity for SAM and SAH by a general lack of interaction between the RNA and the aminocarboxypropyl side chains of these effectors (Huang et al, 2020; Weickhmann et al, 2019). This is reminiscent of Cbl-riboswitches which similarly have few molecular contacts between the RNA and corrinoid tail (Gallo et al, 2011; Gallo et al, 2008; Johnson et al, 2012; Peselis and Serganov, 2013).…”

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN): The Present and Future of the Burgeoning Field

“…Further investigations into the structure, folding, and activity of RNAs have been carried out by the Lilley/Norman Lab at the University of Dundee, where recent studies have looked at the structural mechanisms of dynamic systems such as the pistol and hammerhead ribozymes and the S -adenosylmethionine (SAM)/ S -adenosylhomocysteine (SAH) riboswitch . They have also characterized noncoding Box C/D RNA–protein complexes, which function as independent motifs …”

“…Further investigations into the structure, folding, and activity of RNAs have been carried out by the Lilley/Norman Lab at the University of Dundee, where recent studies have looked at the structural mechanisms of dynamic systems such as the pistol and hammerhead ribozymes 36 and the S-adenosylmethionine (SAM)/S-adenosylhomocysteine (SAH) riboswitch. 37 They have also characterized noncoding Box C/D RNA− protein complexes, which function as independent motifs. 38 The final talk on structural studies was from the Shi-Jie Chen research group at the University of Missouri, Columbia, where computational work has led to the recent development of a Web server, the VfoldLA server, for the prediction of RNA 3D structures.…”

NMR-based structural and functional studies of RNAs and their complexes

“…Lilley and coworkers also showed in FRET experiments that the riboswitch exists in three distinct forms: an unbound and/or flexible population, a compact structure, and a dynamic population (Huang et al 2020). The equilibrium is shifted to the compact structure in presence of ligand.…”

“…Recently, Lilley and coworkers (Huang et al 2020) solved the crystal structure of the SAM/SAH binding riboswitch in complex with SAH, SAM, and other related analogues, respectively. In the following, the numbering according to Publications 3 and 4 will be used.…”