Figure 5.

Schematic of PreQ₁-I riboswitch folding pathway (A) (i) The Bsu riboswitch initially folds into a hairpin followed by a flexible ssRNA tail. (ii) The ssRNA tail, in the presence of divalent cations, dynamically samples a variety of conformations (illustrated in gray), some of which bring L3 close to P1. (iii) On addition of PreQ₁, the P2 stem forms and L3 docks into the P1 minor groove. L1 and L2 show dynamics on the µs-ms and ps-ns timescales, respectively (open arrows). (iv) Divalent cations bind to L1, quenching local flexibility while retaining L2 motions. (B) Alternate P1A hairpin observed in Bsu NMR construct as a result of additional 5’ GG residues. (C) A palindromic sequence commonly observed overlapping with the P2 site in the P1 loop gives rise to dimers. (D) Effect of Bsu loop mutagenesis on apparent PreQ₁ binding. +++ better than WT, ++ equivalent to WT, + and +/− worse than WT, − no detectable binding. Multiple mutations or mutations to L3 are inset. Mutations in Bsu L2var are shown in gray.