Figure 2

Incorporation and misincorporation by wild-type (WT) and mutant RNA polymerase (RNAPs). (a) Cartoon schematically describes the reaction of cNTP (cGTP) incorporation in EC^G1 (Additional File1: Figure S2) with ³²P 5'-labelled RNA (asterisk). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Representative gels of 100 μM cGTP incorporation by WT and 500 μM cGTP incorporation by ΔTL in EC^G1 are shown. The lack of complete extension of transcripts was due to the procedure by which elongation complexes were assembled (see Methods). (b) The intrinsic proofreading reaction accompanies misincorporation. The cartoon above the gel schematically describes the processes going on during ncGTP misincorporation in EC^A (Additional File 1: Figure S2). Elongation complexes are shown with non-template DNA strand below the template strand to reflect their full complementarity (as in Additional File 1: Figure S2). Note that RNAs in elongation complexes were labelled at the 3' end (by incorporation of [α³²P]GTP; asterisk), thus allowing monitoring both misincorporation event and removal of the wrong nucleotide via transcript assisted proofreading. Misincorporation of 1 mM ncGTP and proofreading by WT, H1242A and R1239A RNAPs are shown as an example. The cleavage products larger than dinucleotide originate from 2 bp and 3 bp backtracked complexes that undergone further extension after misincorporation. The colours of the RNA products of the reactions are the same as in the scheme of the reaction above the gels. Black vertical line separates lanes originating from the same gel that were brought together.