Fig. 5: Biochemical characterization of CF promoter recognition.

a ExoIII footprinting assay performed on the dsDNA transcription scaffold (−50 to +20) labelled either on the template or non-template strand. Lanes from left to right: M1, marker for template strand; C, the transcription scaffold without ExoIII as a control; WT, wild-type (wt) transcription scaffold labelled on the template strand ± CF; C, the wt transcription scaffold labelled on the non-template strand without ExoIII as a control; samples of ±CF, the block exchange 1 (BE1) labelled on the non-template strand ±CF, the block exchange 2 (BE2) labelled on the non-template strand ±CF, the −27 to −23 mutant labelled on the non-template strand ±CF, the −22 to −19 mutant labelled on the non-template strand ±CF, M2, marker for the mutant scaffolds. Arrows point to the band resulting from CF binding on the wt template strand (dark blue), wt non-template strand (cyan), BE1 non-template strand (green) and BE2 non-template strand (purple). The cartoon of transcription scaffolds used for footprinting is shown as lines and the specificity box as circles; BE mutants are shown by green and purple lines. b Promoter-dependent in vitro transcription assay was performed using the wt transcription scaffold as in the cryo-EM sample. Top panel: The lanes from left to right show: sample without Rrn3, sample without CF, WT transcription scaffold, BE1, BE2, −29 to −27 mutant, −27 to −23 mutant, −27 mutant, −27 to −26 mutant, −25 mutant, −24 to −23 mutant, −22 to −19 mutant, −14 to −12 mutant, −11 to −7 mutant. Middle panel: The lanes from left to right show: sample without CF, WT transcription scaffold, CF with Rrn7 residues 291–294 mutated to alanine, CF with Rrn7 residues 291–294 DERH mutated to RHDE, CF with Rrn11 with residues R11, K12, K16 and K18 mutated to alanine, CF with Rrn11 with residues R11, K12, K16 and K18 mutated to glutamate, CF with Rrn6 ΔCterm (Δ776–894), sample without Rrn3, Rrn3 Δacidic loop (Δ253–319). Bottom panel: Schematic representation of the template strand of the promoter sequence from −29 to −7 is shown as blue circles. The mutated nucleotides are indicated with the same colour as in the transcription assay. Source data are provided as a Source Data file.