Fig. 1: Overview of the TECdisplay procedure.

a General TECdisplay workflow. A library of RNA variants is encoded in template DNA that contains a promoter that is compatible with dU-excision tagging and a transcription stall site that prevents RNAP from running off the template. Single-round transcription is performed under conditions that yield TECs with a 1:1:1 DNA:RNAP:RNA composition. The TEC library is then fractionated by RNA function. This fractionation step can vary depending on the RNA function being measured. The protein and RNA components of the transcription reaction are then degraded and fraction-specific barcodes are quantitatively appended to template DNA using the one-pot dU-excision tagging procedure. Barcoded fractions are pooled together, the non-transcribed DNA strand is degraded by lambda exonuclease, and the resulting ssDNA is amplified for ~7 cycles of PCR and sequenced to determine the distribution of template DNA across fractions for each variant in the library. Because DNA was fractionated by RNA function, the distribution of DNA between fractions reflects RNA function. b Strategy for measuring riboswitch-mediated transcription termination/antitermination activity using TECdisplay. TECs are synchronized using the C3-SC1 leader sequence and immobilized on streptavidin-coated magnetic beads by a biotinylated capture oligonucleotide. An etheno-dA transcription stall site is positioned downstream of the riboswitch intrinsic terminator to retain RNAP on DNA. When transcription is resumed, template DNA molecules on which a termination event happened are released into the supernatant. Conversely, template DNA molecules on which a terminator readthrough event happened are retained in the bead pellet. c Quantification of C. beijerinckii pfl ZTP riboswitch activity in various conditions using the fractionation strategy shown in panel b. Fraction terminator readthrough was calculated using the distribution of template DNA between supernatant and pellet fractions and using the distribution of terminated and full-length RNA for each sample. The gel image is representative of n = 2 replicates. Uncropped source gels are available in Supplementary Fig. 9. Source data are provided as a Source Data file. RNAP RNA polymerase, C3-SC1 Cap3-structure cassette 1.