Fig. 1.

Structure-guided design of a TPP Spinach riboswitch. (A) Metabolite-dependent natural switching mechanism of TPP riboswitches. Riboswitches contain an aptamer domain (blue) and an expression platform (black). In the absence of TPP binding, the transducer sequence (gray) is hybridized to the switching sequence (yellow) in the aptamer domain, leaving the SD sequence accessible. When TPP binds, the aptamer domain undergoes a structural rearrangement, which disrupts this strand hybridization. The transducer sequence is displaced from the switching sequence and hybridizes to the SD sequence, forming the helical structure of the expression domain. (B) Secondary structure and design of TPP Spinach riboswitch. The Spinach riboswitch is generated by replacing the expression platform of the thiM TPP riboswitch with Spinach (green). TPP binding releases the transducer sequence (gray) from the switching sequence (yellow), forming a critical helix in Spinach. Formation of this helix is critical for Spinach to bind and activate the fluorescence of DFHBI (green ball). A linker sequence (dark gray) is used to position the aptamer domain and Spinach optimally. (C) Structural model of DFHBI binding to Spinach. The figure was prepared with PyMOL software based on previously reported Spinach structure (29). The portion of Spinach that contains the transducer sequence that is modified to form the TPP Spinach riboswitch is shown. The structural model highlights the DFHBI-binding pocket (green ball-and-stick model), which is formed by a U⋅A⋅U base triple (teal; bases are labeled with letters) and a G-quadruplex (deep violet). The portion of the H₁ helix (5′-UCCA-3′) that provides the U in the U⋅A⋅U base triple is indicated in light gray. This sequence is modified to form the transducer sequence in the TPP Spinach riboswitch.