Fig. 4: NTPase activities of HelD and release of HelD from RNAP.

a, b Comparison of NTPase activities of free HelD and its complexes with RNAP. ATP/GTP hydrolyzing activity of free HelD was set as 100%. ATP hydrolysis (a) is stimulated upon complex formation, whereas GTP hydrolysis (b) remains almost unchanged. Control measurements for individual complex components are shown. The bars show averages from three biological replicates, the error bars are ±SD, the dots represent individual experiments (also in panels d–g). c A scheme depicting the HelD release assay: His–RNAP was reconstituted into three different complexes, each containing combinations of HelD (cayn), σ^A (purple) and RbpA (yellow). The RNAP complexes were then allowed to bind to magnetic beads. The amount of HelD released, with or without addition of other factors (in panels d–g) was determined by Coomassie blue-stained SDS-PAGE gels and densitometry. d Effect of 1 mM ATP, GTP, or CTP on HelD release. In panels d–g, representative primary data are shown above the graph. Zero (Ø) shows HelD release without the addition of other factors. For this and experiments (e, g), the His–RNAP complex containing HelD, σ^A, and RbpA was used (depicted within the dashed box in c). The amount of HelD released from RNAP–σ^A–RbpA–HelD by the addition of ATP was set as 1 (also in other panels). A second primary data example is shown in Supplementary Fig. 14 together with a calibration curve used as quantification control. e, Effect of ATP analogs on HelD release. RNAP complexes were reconstituted as described in panel c with four HelD variants: WT-HelD (wild type), HelD^σA-INT, HelD^A-HYDRO, and HelD^A-BIND (for definition of the mutants see Supplementary Fig. 16). Subsequently, 1 mM each of ATP, N-ATP, or ATPγS was added to the preformed RNAP complex attached to the magnetic beads and release of HelD from the complex was observed. f Release of HelD from the three types of complexes (c) induced with 1 mM ATP. g Effect of two forms of DNA and/or ATP on HelD release. CC, closed complex us-fork promoter DNA. OC open complex DNA with artificially opened transcription bubble. Source data are provided as a Source Data file.