Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in 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 ZTP riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate an internal RNA strand displacement process and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for pfl riboswitch antitermination and suggest general strategies by which nascent RNA molecules navigate cotranscriptional folding pathways.

No 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.

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.

Genetically modifying T cells can enable applications ranging from cancer immunotherapy to HIV treatment, yet delivery of T cell-targeted therapeutics remains challenging. Extracellular vesicles (EVs) are nanoscale particles secreted by all cells that naturally encapsulate and transfer proteins and nucleic acids, making them an attractive and clinically-relevant platform for engineering biocompatible delivery vehicles. We report a suite of technologies for genetically engineering cells to produce multifunctional EV vehicles—without employing chemical modifications that complicate biomanufacturing. We display high affinity targeting domains on the EV surface to achieve specific, efficient binding to T cells, identify a protein tag to confer active cargo loading into EVs, and display fusogenic glycoproteins to increase EV uptake and fusion with recipient cells. We demonstrate integration of these technologies by delivering Cas9-sgRNA complexes to edit primary human T cells. These approaches could enable targeting vesicles to a range of cells for the efficient delivery of cargo.