Cotranscriptional RNA strand exchange underlies the gene regulation mechanism in a purine-sensing transcriptional riboswitch

Abstract

RNA folds cotranscriptionally to traverse out-of-equilibrium intermediate structures that are important for RNA function in the context of gene regulation. To investigate this process, here we study the structure and function of the Bacillus subtilis yxjA purine riboswitch, a transcriptional riboswitch that downregulates a nucleoside transporter in response to binding guanine. Although the aptamer and expression platform domain sequences of the yxjA riboswitch do not completely overlap, we hypothesized that a strand exchange process triggers its structural switching in response to ligand binding. In vivo fluorescence assays, structural chemical probing data, and experimentally informed secondary structure modeling suggest the presence of a nascent intermediate central helix. The formation of this central helix in the absence of ligand appears to compete with both the aptamer's P1 helix and the expression platform's transcriptional terminator. All-atom molecular dynamics simulations support the hypothesis that ligand binding stabilizes the aptamer P1 helix against central helix strand invasion, thus allowing the terminator to form. These results present a potential model mechanism to explain how ligand binding can induce downstream conformational changes by influencing local strand displacement processes of intermediate folds that could be at play in multiple riboswitch classes.

Cotranscriptional RNA strand displacement underlies the regulatory function of the E. coli thiB TPP translational riboswitch

“…Taken together, these results support the importance of pairing between the 5’ and 3’ sides of P1 and the 3’ side of P1 with the linker to form an anti-sequestering hairpin for riboswitch switching. These results match those seen in other riboswitch systems thought to utilize strand displacement to form intermediate structures in their switching mechanism (Cheng et al 2022), strongly suggesting a similar strand displacement mechanism is present in the E. coli thiB mechanism.…”

“…This same study found lengthening the P1 helix by 2 GC basepairs stabilized P3/L5 interactions, which could potentially contribute to understanding how TPP binding prevents strand displacement (Haller et al 2013). While not tested here, this leads to a possible second event that could tune riboswitch function – as the anti-sequester stem competes with the P1 hairpin, the sequestering stem can also begin to nascently form downstream, creating a competition between anti-sequestering and sequestering stem formation that could ultimately tune riboswitch function as has been observed in the yxjA transcriptional riboswitch (Cheng et al 2022).…”

“…In addition, this linker region sequence is identical to the 5’ side of the P1 stem (nts 5-9), suggesting that the P1 helix and the anti-sequestering helix could be mutually exclusively basepaired regions within the riboswitch. Notably, such internal competing structures have been identified in other riboswitch mechanisms that appear to leverage strand displacement in their switching mechanisms (Strobel et al 2019; Cheng et al 2022).…”

“…Previous work has shown that sequences directly after the P1 helix can play a role in riboswitch mechanisms through the formation of intermediate structures that compete with EP folding to enact the switch (Cheng et al 2022). Consequently, we next sought to determine whether this sequence could play a role in the thiB switching mechanism.…”

“…One mechanism that allows RNAs to traverse free energy barriers to these rearrangements during the time window of transcription is strand displacement (Hong and Šulc 2019), a process by which an invading nucleic acid strand can base pair with a substrate strand by displacing a previously paired substrate:incumbent strand duplex (Hong and Šulc 2019). Strand displacement has been shown to underlie the ability of non-coding RNAs such as the E.coli Signal Recognition Particle to cotranscriptionally rearrange into long-range structures (Yu et al 2021; Fukuda et al 2020), as well provide a mechanism by which EPs can displace apo-ADs in diverse transcriptional riboswitches including those that sense ZTP (Strobel et al 2019; Hua et al 2020), fluoride (Watters et al 2016), and guanine (Cheng et al 2022). The formation of a ligand-bound holo-AD is then understood to block strand displacement, allowing transcriptional riboswitches to govern EP folding and gene regulation through AD-ligand interactions.…”