Sequence-dependent folding and unfolding of ligand-bound purine riboswitches.
Riboswitch regulation of gene expression requires ligand-mediated RNA folding. From the fluorescence lifetime distribution of bound 2-aminopurine ligand, we resolve three RNA conformers (C(o), C(i), C(c)) of the liganded G- and A-sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg(2+), together with results from mutagenesis, suggest that C(o) and C(i) are partially unfolded species compromised in key loop-loop interactions present in the fully folded C(c). These data verify that the ligand-bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand-mediated folding of the G-sensing riboswitch is more effective, less dependent on Mg(2+), and less debilitated by mutation, than the A-sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence-dependent RNA dynamics, which adjust the balance of ligand-mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine-tuning of gene regulatory mechanisms.
Prychyna
Oksana
O
Department of Chemistry, Simon Fraser University, Burnaby, BC, Canada, V5A 1S6.
Dahabieh
Michael S
MS
Chao
Jay
J
O'Neill
Melanie A
MA
D020121:5' Untranslated Regions / Q000235:genetics; D052157:Aptamers, Nucleotide / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics; D001483:Base Sequence; D005798:Genes, Bacterial; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D011687:Purines / Q000378:metabolism*; D012329:RNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014534:UDPglucose 4-Epimerase / Q000235:genetics
D020121:5' Untranslated Regions; D052157:Aptamers, Nucleotide; D008024:Ligands; D011687:Purines; D012329:RNA, Bacterial; D012333:RNA, Messenger; D014534:UDPglucose 4-Epimerase