Energy Landscape Analysis of the Full-Length SAM-I Riboswitch using Single-Molecule FRET Spectroscopy

Abstract

Binding of the ligand S-adenosyl-L-methionine (SAM) produces major structural changes in the SAM-I riboswitch (RS) and thereby regulates gene expression via transcription termination. As yet, the conformations and motions governing the function of the full-length Bacillus subtilis yitJ SAM-I RS have not been deeply investigated. We have studied its conformational energy landscape as a function of Mg2+ and SAM ligand concentrations using single-molecule Förster resonance energy transfer (smFRET) microscopy. smFRET histograms of differently FRET-labeled constructs were so complicated that they could only be resolved with the help of kinetic experiments on immobilized riboswitches and hidden Markov modeling (HMM) analysis. At least four conformational states were identified, both in the presence and the absence of SAM. We determined their Mg2+-dependent fractional populations and conformational dynamics, including state lifetimes, interconversion rate coefficients and equilibration timescales. Riboswitches with terminator and antiterminator folds were found to coexist under all conditions; SAM binding induced only a gradual increase in the population of terminator states. Conformational transitions were much faster with bound SAM, which may be crucial for off-switching during the brief decision window before expression of the downstream gene.