Fig. 1: RPo formation kinetics of EσN informs cryo-EM analysis.
a Stopped-flow measurement employs a linear, Cy3-labeled (yellow) σN promoter DNA (Aae dhsU). The fluorophore is subject to protein-induced fluorescent enhancement (PIFE) when RPo forms. For the experiment, an early-melted intermediate (box 1; E + σN+dhsU+C1) is pre-assembled from core RNAP (E, gray), σN (orange), and Cy3-labeled dhsU (green) in presence of bEBP (C1; blue). ATP is prepared in buffer (box 2). Mixing the contents of boxes 1 and 2 will start the reaction and PIFE can be observed. b RPo formation-dependent fluorescence saturates at around 8–10 min reaction time and is dependent on the presence of all components (black; n = 6). Mixing ATP with complexes without C1 (red; n = 5), ATP with DNA alone (blue; n = 3), or the E + σN+dhsU+C1 with buffer (no ATP, green; n = 6) results in only background signal. Solid lines represent the average of the individual mixing events. Dotted lines indicate error bands of one standard deviation. The arrow indicates the time point used for cryo-EM sample preparation. c Cryo-EM analysis of samples prepared after 35 s reaction time yields three major populations: early-melted intermediates (RPem, 31% of particles), bEBP-bound complexes (55% of particles), and complexes with a melted promoter DNA (RPo-like, 14%). Maps from the 3D classification (see main text and methods for details) were colored according to core RNAP (gray), σN (orange), dhsU (green), and bEBP (blue). The dashed arrows indicate the sequence of the complexes along the transcription initiation pathway. Source data are provided as a Source Data file.
