Abstract
RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. To investigate how these processes jointly modulate the function of the folate stress-sensing Fusobacterium ulcerans ZTP riboswitch, we apply a single-molecule vectorial folding (VF) assay in which an engineered superhelicase Rep-X sequentially releases fluorescently labeled riboswitch RNA from a heteroduplex in a 5′-to-3′ direction, at ~60 nt s−1 [comparable to the speed of bacterial RNA polymerase (RNAP)]. We demonstrate that the ZTP riboswitch is kinetically controlled and that its activation is favored by slower unwinding, strategic pausing between but not before key folding elements, or a weakened transcription terminator. Real-time single-molecule monitoring captures folding riboswitches in multiple states, including an intermediate responsible for delayed terminator formation. These results show how individual nascent RNAs occupy distinct channels within the folding landscape that controls the fate of the riboswitch.
Highlights
RNAs begin to fold and function during transcription
The F. ulcerans ZTP riboswitch regulates gene expression by binding to 5aminoimidazole-4-carboxamide riboside 5′-monophosphate and triphosphate (ZMP and ZTP, respectively), which are elevated during folate stress[4,5,6]
ZMP binding to the ZTP riboswitch aptamer is sensitive to 3′-end extension after nt 75, and variants containing as few as five base pairs of the terminator bind poorly to ZMP (Supplementary Fig. 1), suggesting that even partial termination hairpin folding competes with ligand binding
Summary
RNAs begin to fold and function during transcription. Riboswitches undergo cotranscriptional switching in the context of transcription elongation, RNA folding, and ligand binding. As gene expression regulatory elements, riboswitches are structurally divided into two domains—an aptamer domain sufficient for ligand binding and an expression platform regulating gene expression[1,2,3] These two domains share an overlapping sequence making their formation mutually exclusive. Investigations of the kinetic control model are often preceded by the observation of a discrepancy between equilibrium ligandbinding measurements and cotranscriptional riboswitch activation measurements, suggestive of a non-equilibrium (kinetically controlled) process This discrepancy is further examined through monitoring ligand binding and RNA folding kinetics. Chemical probing approaches were combined with high-throughput sequencing and used to examine riboswitch structure during roadblocked transcription to obtain more complete maps of RNA folding for all transcript lengths[13,14]. Both a drawback and an advantage of the optical tweezer assay are that tension is applied to either the nucleic acid substrates or RNAP, examining force-dependent perturbation of the system
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