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. 2003 Nov 15;17(22):2839–2851. doi: 10.1101/gad.1142203

Figure 3.

Figure 3.

(A) In vitro transcription assay. β′::σ70 or wild-type RNAPs (40 nM) were allowed to initiate transcription on a linear DNA template (25 nM) with ApU, ATP, [32P]CTP, and GTP. Samples were mixed with 2× STOP buffer at 10, 20, 30, 45, 60, 75, 90, and 120 sec. UTP was then added to allow transcription past A29, and additional samples were taken 10, 20, 30, 45, 60, 120, 240, and 480 sec later (final sample 10 min after reaction started). The samples were separated by 15% denaturing PAGE. (AUC) Trinucleotide abortive product; (A29) A29 EC; (P) his pause RNA; (RO) run-off RNA. (B) Kinetics of promoter association, following the mechanism defined by Saecker et al. (2002). kf,obs and kr,obs were obtained for β′::σ70 and wild-type RNAPs on the template shown in panel A (≥3 independent experiments; Materials and Methods).