Abstract
Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure and function by forming transient intermediate folds. This is especially true for transcriptional riboswitches in the which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways and the mechanisms that enable transit between them remain largely unknown. Here we determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate a regulatory RNA strand displacement reaction and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for ZTP riboswitch antitermination and suggest general strategies by which nascent RNA molecules can navigate cotranscriptional folding pathways efficiently.