Extended Data Fig. 8: DNA endonuclease activity of RNAP at the ThreeSSeq consensus sequence, the rpoB2-sensitive component.
From: Endogenous DNA damage at sites of terminated transcripts
a, Transcription complexes were assembled with E. coli core RNAP on nucleic acid scaffolds composed of template DNA (T DNA, gray), non-template DNA (NT DNA, blue), and RNA (red). The RNA, which anneals to the T DNA strand, defines the geometry of the RNAP-DNA complex. In the absence of RNA, RNAP binds in either of two orientations: binding either the blue or gray DNA strand as template. In panel b, only one scaffold orientation is shown: blue strand on top. But in all DNA substrates with neither protein nor RNA bound, RNAP binds either top or bottom strand as template. b, Indicated DNA strands were labeled at the 5’ end with γ32P-ATP and T4 PNK (label position shown by a yellow star). Assembled complexes were incubated at 37 °C for 15, 30, and 60 min; E. coli RfaH, which specifically binds to the GCGGTAGC element in the NT DNA, was present where shown. After quenching, DNA products were analysed on a 10% urea-acrylamide gel and visualized by phosphorimaging. A dashed line indicates samples from different gels. DNA ladder was made by mixing DNA oligonucleotides of indicated lengths and labeling the mixture with γ32P-ATP and T4 PNK; the 30-mer was present in 3x molar excess. The red X indicates the position of two apparent cleavage products in the non-template DNA; the resulting products migrate as 26- and 27-mers. Each experiment was performed three times with similar results. Primary data are provided in the Source Data File. Two negative controls indicate consensus-sequence specificity of the cleavage: (i) the 5’- labeled T-DNA, which would show cleavage in the bottom gray strand if cleavage were non-specific and could occur next to the 5’ACCCGCGT sequence there; and (ii) the 5’-labeled NT AA DNA, which carries the 5’AATTTTTT sequence in place of the 5’CCTTTTTT consensus, at the same position, with same strand labeled. Neither of these substrates elicits cleavage 5′ of those sequences as is seen with the active consensus sequence in labeled strand (leftmost, 5’-labeled NT-CC DNA). For gel source data, see Supplementary Fig. 1.
