title: Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy.
abstract: The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Ruff
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8114, USA scott.strobel@yale.edu.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Strobel
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D005998:Glycine / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism; D058928:Riboswitch
chemical_list: D052157:Aptamers, Nucleotide; D012329:RNA, Bacterial; D012333:RNA, Messenger; D058928:Riboswitch; D005998:Glycine
keywords: aptamers; dimerization; glycine; riboswitch; tandem