Figure 6.

Model of S_MK box structural transitions in response to SAM. (a) Observed conformations of S_MK box. Left, the free state observed in SAM-free S_MK59 via NMR, with accessible SD sequence. Center, the poorly-folded state observed via NMR in SAM-free S_MK51 (which is missing the eight 5’ nucleotides shown here in grey), with a partially-accessible SD sequence. Right, the well-folded SAM-bound state with occluded SD sequence, observed in ligand-bound S_MK51 and S_MK59 and the previously-determined crystal structure. Hydrogen bonds are represented as lines as observed via NMR for ISO_SMK and PRIMED_SMK, while hydrogen bonds expected based on the crystal structure¹⁰ are shown for BOUND_SMK. Dashed lines in the PRIMED_SMK state represent transiently formed base pairs as detected via NMR. (b) A schematic representing the transitions observed in ITC for S_MK51 or S_MK59 and how the analogous constructs behave while undergoing similar transitions inside the cell, as measured by β-galactosidase reporter assays. (c) Effect of the alternatively folded conformation on the populations of riboswitch conformations. SAM dependence of molecular species are simulated from equations (2, 3) describing the fractional populations of the SAM-bound species (S_MK51_BOUND, and S_MK59_BOUND) and of the effective concentration of exposed SD sequence for each construct. Curves were generated with the experimentally determined values of K_A and K_ISO, and using a relative accessibility of the SD in the PRIMED_SMK form, α, estimated to be 0.1. The equilibrium between the ISO_SMK and PRIMED_SMK conformations (1) shifts the affinity of the riboswitch into the biologically-relevant micromolar range, and (2) amplifies the response of the switch to SAM by promoting greater SD accessibility at low [SAM].