Nonlocal helix formation is key to understanding S-adenosylmethionine-1 riboswitch function

Abstract

Riboswitches are noncoding RNAs that regulate gene expression in response to changing concentrations of specific metabolites. Switching activity is affected by the interplay between the aptamer domain and expression platform of the riboswitch. The aptamer domain binds the metabolite, locking the riboswitch in a ligand-bound conformation. In absence of the metabolite, the expression platform forms an alternative secondary structure by sequestering the 3' end of a nonlocal helix called P1. We use all-atom structure-based simulations to characterize the folding, unfolding, and metabolite binding of the aptamer domain of the S-adenosylmethionine-1 (SAM-1) riboswitch. Our results suggest that folding of the nonlocal helix (P1) is rate-limiting in aptamer domain formation. Interestingly, SAM assists folding of the P1 helix by reducing the associated free energy barrier. Because the 3' end of the P1 helix is sequestered by an alternative helix in the absence of metabolites, this observed ligand-control of P1 formation provides a mechanistic explanation of expression platform regulation.

Figure 1

(a) Secondary and (b) tertiary structure (PDB entry: 2GIS) of the SAM-I riboswitch. Average secondary structure formation as a function of the fraction of native contacts formed (Q; see the Supporting Material) for the (c) SAM-free and (d) SAM-present simulations. (a–d) Color scheme: P1, cyan; P2, red; P3, green; P4, blue; PK, orange; and SAM, purple in panels b and d. In panel a, SAM-contacting residues are highlighted by brown boxes. The most notable difference in folding mechanism is earlier initial folding of P1 (black arrows) at the expense of the PK (starred) when SAM is present. The folding free-energy profiles for the (e) SAM-free and (f) SAM-present simulations are shown for several temperatures (with temperature indicated by color). The most significant free-energy barrier in both systems is associated with initial P1 folding. When SAM is present, the free-energy barrier is reduced and encountered earlier in the folding process.

Figure 2

(a) Average percent of SAM-aptamer domain interactions formed by region as a function of the fraction of native SAM-aptamer domain contacts formed Q_SAM. Simulation images illustrating the SAM binding mechanism: (b) SAM binds a preformed P3 helix; (c) SAM recruits 3′ strand of P1; (d) SAM binds 5′ strand of P1; and P1 helix formation proceeds.