Abstract / Summary
Riboswitches are comparatively simple regulatory RNA devices that afford ligand control of gene expression.
We have studied structural and dynamic properties of the SAM riboswitch in response to its cognate ligand S-adenosyl-L-methionine (SAM) and its derivative S-adenosyl-L-homocysteine (SAH) by using advanced single molecule-based fluorescence methods based on Förster resonance energy transfer. Six riboswitch constructs were synthesized that carried donor and acceptor dyes at different attachment sites. The fluorescence emission from the FRET pair labeled constructs was measured and analyzed with respect to intensity of the donor and acceptor emission to identify differently folded conformations on the basis of FRET in the ligand-free construct. Mg2+, SAM and SAH titration experiments were carried out on freely diffusing RNA molecules to determine the respective affinities from FRET efficiency variations. Moreover, conformational changes upon ligand binding were monitored in real time on surface-immobilized riboswitch preparations.