Abstract
The thiamine pyrophosphate (TPP) riboswitch is a cis-regulatory element in mRNA that modifies gene expression in response to TPP concentration. Its specificity is dependent upon conformational changes that take place within its aptamer domain. Here, the role of tertiary interactions in ligand binding was studied at the single-molecule level by combined force spectroscopy and Förster resonance energy transfer (smFRET), using an optical trap equipped for simultaneous smFRET. The 'Force-FRET' approach directly probes secondary and tertiary structural changes during folding, including events associated with binding. Concurrent transitions observed in smFRET signals and RNA extension revealed differences in helix-arm orientation between two previously-identified ligand-binding states that had been undetectable by spectroscopy alone. Our results show that the weaker binding state is able to bind to TPP, but is unable to form a tertiary docking interaction that completes the binding process. Long-range tertiary interactions stabilize global riboswitch structure and confer increased ligand specificity.
Highlights
The gene-regulatory activity of a riboswitch is mediated by its ability to form a substructure, called the aptamer domain, that binds—and thereby senses—a ligand, which is generally a small metabolite (Roth and Breaker, 2009; Serganov and Nudler, 2013; Serganov and Patel, 2012)
Riboswitch aptamers fold and bind their cognate ligands in a highly specific manner, and as they do so, they compete with alternative RNA structures that can form in conjunction with other domains of the riboswitch, which function either to permit, or to prevent, downstream gene expression
Tertiary interactions were simultaneously scored by a Forster resonance energy transfer (FRET) readout between donor and acceptor fluorescent dyes, ATTO550 and ATTO647N (Figure 1B), which were covalently attached to the L5 and P3 helix arms, respectively
Summary
The gene-regulatory activity of a riboswitch is mediated by its ability to form a substructure, called the aptamer domain, that binds—and thereby senses—a ligand, which is generally a small metabolite (Roth and Breaker, 2009; Serganov and Nudler, 2013; Serganov and Patel, 2012). Crystal studies have identified striking similarities between the structures of the ligand-bound forms of a truncated TPP aptamer from A. thaliana and the thiM aptamer from Escherichia coli (Edwards and Ferre-D’Amare, 2006; Serganov et al, 2006; Thore et al, 2006). Both eukaryotic and prokaryotic aptamers share a common binding conformation (Figure 1), Duesterberg et al eLife 2015;4:e12362.
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