Fig. 1: Scaffold design, reconstitution strategy, and evaluation for the putEC.

a The nucleic acid scaffold in the reconstituted putEC. The DNA was synthesized by PCR, and the RNA is synthesized in vitro from Eco RNAP holoenzyme by adding rNTP as described in b. The transcription regulation elements including −35 element, −10 element, putRNA, −10-like sequence, the pausing site, and lacO sequence for roadblocking are shown. Transcription bubble is marked with blue dotted box. Transcript numbering is written for reference. b A schematic diagram of the putEC reconstitution. First, Eco holoenzyme containing Eco RNAP and σ⁷⁰ binds to the promoter region of the DNA forming RPo. LacI is added to the RPo to make a roadblock at the lacO site and rNTP is added to initiate the transcription. After transcribing putRNA, the RNAP stalls due to the roadblocking LacI. Then, free rNTP is removed by using size-exclusion column before LacI is detached by IPTG to prevent further RNA synthesis. The prepared, active putEC is used for cryo-EM and nMS analyses. c Radiolabeled transcription assay reveals that the wild-type (WT) put sequence (from pRAK31¹¹) did not display paused transcript at the pausing site (lane 2) while the put⁻ sequence showed the paused transcript band (lane 3). The designed DNA scaffold, 7-nt, did not show pausing in the absence of LacI, but stalled at the pausing site when LacI was added (Lane 4, 5). For the lanes 2–5, the transcription reaction was done for 2 min at 37 °C. To show the roadblocked EC is still capable of transcription, we added 2 mM IPTG, incubated for 2 min, and resumed the transcription by adding additional rNTP. The resumed reaction mixture was quenched after 2 min and loaded onto the gel (Lane 6, labeled ‘Resume’). Details are in the Method section. RO stands for run-off. d The RNA extracted from the reconstituted putEC is analyzed by nMS. The result revealed two main peaks of C94 and U95, labeled as 94-mer and 95-mer, respectively. The ratio between 94-mer and 95-mer was approximately 2:3. Partial run-off products were observed at higher mass region. Source data are available as a Source Data file.