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.

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.

Cotranscriptional RNA folding forms structural intermediates that can be critically important for RNA biogenesis. This is especially true for transcriptional riboswitches that must undergo ligand-dependent structural changes during transcription to regulate the synthesis of downstream genes. Here, we systematically map the folding states traversed by the Clostridium beijerinckii pfl riboswitch as it controls transcription termination in response to the purine biosynthetic intermediate ZMP. We find that after rearrangement of a non-native hairpin to form the ZTP aptamer, cotranscriptional ZMP binding stabilizes two structural elements that lead to antitermination by tuning the efficiency of terminator hairpin nucleation and strand displacement. We also uncover biases within natural ZTP riboswitch sequences that could avoid misfolded intermediates that disrupt function. Our findings establish a mechanism for ZTP riboswitch control of transcription that has similarities to the mechanisms of diverse riboswitches and provide evidence of selective pressure at the level of cotranscriptional RNA folding pathways.superfolder GFP (SFGFP) coding sequence, listed as 'trailing sequence' below. Promoter sequences are blue. Mutation positions are highlighted with red. Deletion positions are shown as "-". Description Sequence Promoter GCTTGATTCTAAAGATCTTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGT C. beijericnckii ZTP pfl riboswitch ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGTCTGGGCAAAAAAAGCCTGGATTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch PK4 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGATCGCCTGGGCAAAAAAAGCCTGGGTTGCATCGGCTTTTTTAT Cbe ZTP riboswitch PK5 ATATTAGATATTAGTCATATGACTGGCGAAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGATCGCCTGGGCAAAAAAAGCCTGGGTTGCATCGGCTTTTTTAT Cbe ZTP riboswitch PKAT ATATTAGATATTAGTCATATGACTGACGAAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGATCGTCTGGGCAAAAAAAGCCTGGATTGCATCGGCTTTTTTAT Cbe ZTP riboswitch PKGC ATATTAGATATTAGTCATATGACTGGCGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGCCTGGGCAAAAAAAGCCTGGGTTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch PKMM ATATTAGATATTAGTCATATGACTGACGAAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGCCTGGGCAAAAAAAGCCTGGGTTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch T1 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGTCTGGGCAAAAAAAGCCCGGATTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch T2 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGTCTGGGCAAAAAAAGCCTGGA-TG-GTCGGCTTTTTTAT Cbe ZTP riboswitch T3 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGTCTGGGCAAAAAAAGCCTGGA-CG-GTCGGCTTTTTTAT Cbe ZTP riboswitch H1 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAATCGCCGACCGTCTGGGCGCAAAAAAAGCGCCTGGATTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch H2 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCAT...

Riboswitches are cis-regulatory RNA elements that regulate gene expression in response to ligand binding 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 structural intermediates that compete with the AD and EP folds to mediate the switching mechanism on the timescale of transcription. Here we investigate whether similar intermediates are important for riboswitches that control translation 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. Sequence complementarity between the linker region and the AD P1 stem suggested the possibility of an intermediate nascent RNA structure called the anti-sequestering stem that could mediates the thiB switching mechanism. Experimentally informed secondary structure models of the thiB folding pathway generated from chemical probing of nascent thiB structures in stalled transcription elongation complexes confirmed the presence of the anti-sequestering stem, and showed it may form cotranscriptionally. Additional mutational analysis showed that mutations to the anti-sequestering stem break or bias thiB function according to whether the anti-sequestering stem, or P1 is favored. This work provides an important example of intermediate structures that compete with AD and EP folds to implement riboswitch mechanisms.

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.