Figure 3.

Mfd WA550 displaces stalled transcription ECs and transcription ICs in vitro. ICs were formed in repair buffer by the incubation of RNAP holoenzyme with a radiolabelled DNA fragment (DNA). ECs stalled at +21 (ECs) were formed by the addition of ApU, ATP, CTP, GTP and 3’dUTP. 250 nM WT Mfd or its derivatives and 2 mM dATP were added and samples loaded on an EMSA gel at the time intervals indicated. (A) Graphical representation of time-course reactions monitoring displacement of stalled ECs by Mfd and its derivatives. The ECs were formed with WT RNAP or RNAP βIA117KA118EA119. Data points are expressed as a percentage of the amount of EC at t = 0 and are an average of three independent experiments, with standard deviation. (B–F) Representative images of EMSA gels showing the effect of WT and mutant Mfd proteins on transcription complexes. Asterisk indicates bands of higher electrophoretic mobility which are thought to be due to the presence of two RNAP molecules on the DNA (one of which is bound to the promoter).