Mechanisms for differentiation between cognate and near-cognate ligands by purine riboswitches.
Riboswitches are elements in the 5'-untranslated region of mRNAs that regulate gene expression by directly interacting with metabolites related to their own gene products. A remarkable feature of this gene regulation mechanism is the high specificity of riboswitches for their cognate ligands. In this study, we used a combination of static and time-resolved NMR-spectroscopic methods to investigate the mechanisms for ligand specificity in purine riboswitches. We investigate the xpt-aptamer domain from a guanine-responsive riboswitch and the mfl-aptamer domain from a 2'-deoxyguanosine-responsive riboswitch. The xpt-aptamer binds the purine nucleobases guanine/hypoxanthine with high affinity, but, unexpectedly, also the nucleoside 2'-deoxyguanosine. On the other hand, the mfl-aptamer is highly specific for its cognate ligand 2'-deoxyguanosine, and does not bind purine ligands. We addressed the question of aptamer`s ligand specificity by real-time NMR spectroscopy. Our studies of ligand binding and subsequently induced aptamer folding revealed that the xpt-aptamer discriminates against non-cognate ligands by enhanced life-times of the cognate complex compared with non-cognate complexes, whereas the mfl-aptamer rejects non-cognate ligands at the level of ligand association, employing a kinetic proofreading mechanism.
Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
Anna
A
Wacker
Janina
J
Buck
Christian
C
Richter
Harald
H
Schwalbe
Jens
J
Wöhnert
D052157:Aptamers, Nucleotide / Q000737:chemistry*; D001412:Bacillus subtilis / Q000235:genetics; D003849:Deoxyguanosine / Q000737:chemistry*; D045622:Entomoplasmataceae / Q000235:genetics; D019271:Hypoxanthine / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry*; D058928:Riboswitch
D052157:Aptamers, Nucleotide; D008024:Ligands; D012329:RNA, Bacterial; D058928:Riboswitch; D019271:Hypoxanthine; D003849:Deoxyguanosine