Berman, K. E. | Search bioRxiv

bioRxiv2022

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

Riboswitches are cis-regulatory RNA elements that regulate gene expression in response to ligand through the coordinated action of a ligand-binding aptamer domain (AD) and a downstream expression platform (EP). Previous studies of transcriptional riboswitches have uncovered diverse examples that utilize cotranscriptional strand displacement to mediate the switching mechanism. The coupling of transcription and translation in bacteria motivates the intriguing question as to whether translational riboswitches can utilize the same mechanistic features. Here we investigate this question by studying the Escherichia coli thiB thiamine pyrophosphate (TPP) riboswitch. Using cellular gene expression assays, we first confirmed that the riboswitch acts at the level of translational regulation. Deletion mutagenesis showed the importance of the AD-EP linker sequence for riboswitch function, which based on sequence complementarity with the AD P1 stem suggested the possibility of an intermediate structure reminiscent of transcriptional riboswitches that exploit strand displacement. Point mutation analysis of this intermediate structure, followed by designed changes to P1, supported a strand displacement mechanism for E. coli thiB. This work provides an important new example of diverse riboswitch AD-EP combinations that exploit this switching mechanism.

molecular biology↗

bioRxiv2019

A mechanism for ligand gated strand displacement in ZTP riboswitch transcription regulation

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.

biochemistry↗