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.When a gene is switched on, its DNA is first copied to make a molecule of messenger ribonucleic acid (mRNA). The genetic code in the mRNA is then translated into a protein. There are also untranslated regions within mRNAs that do not code for protein themselves, but serve to regulate whether or not a protein is produced from the rest of the mRNA. For example, many mRNAs contain a motif in their untranslated region called a 'riboswitch'. These motifs selectively bind to molecules that are the products of metabolic processes. One riboswitch found in bacteria, animals and plants binds to a molecule known as thiamine pyrophosphate (TPP) and regulates genes that control the uptake of a vitamin called thiamine into cells.Newly made mRNA molecules are linear strands that then fold into three-dimensional structures. The TPP riboswitch can adopt distinct shapes depending on whether it is bound to TPP or not. Knowledge of these structures is crucial for understanding how riboswitches regulate protein production. Previous research reported the folding of a TPP riboswitch from bacteria.Here, Duesterberg et al. used a combination of two techniques known as force spectroscopy and Förster resonance energy transfer (FRET) to study the folding of the TPP riboswitch from a plant called Arabidopsis thaliana. The experiments show that in the presence of TPP, structural changes occur in two arm-like appendages – known as helix arms – that extend out of the core of the riboswitch. The riboswitch adopts a particular shape when TPP is strongly bound to it, and in this shape the riboswitch can regulate the activity of certain genes. However, if the riboswitch is only weakly associated with TPP, it takes on a shape in which the two helix arms are further apart and the riboswitch is unable to form the interactions required to complete the process of binding to TPP.Duesterberg et al.’s findings reveal that the way in which the A. thaliana riboswitch changes shape when it is bound to TPP is different to that of its bacterial counterpart. The next challenge will be to observe these shape changes in even more detail, and to use these new techniques to study other riboswitches in various organisms.
Duesterberg, V. K., Fischer-Hwang, I. T., Perez, C. F., Hogan, D. W., & Block, S. M. (2015). Observation of long-range tertiary interactions during ligand binding by the TPP riboswitch aptamer. ELife, 4. https://doi.org/10.7554/elife.12362