Fig. 3. The Msm HelD-specific domain interactions with the RNAP primary channel.

a Surface of the Msm RNAP core (PDB ID 6F6W), color-coded as in Fig. 1a with the description of individual domains and functional parts. b, c Surface representation of States I and II of the Msm HelD–RNAP complex with RNAP color-coding as in a and marked domain names; HelD color-coding as in Fig. 1e. d, e Ribbon representation of the HelD-specific domain inserting into the RNAP primary channel in State I (d) and State II (e). In State I (d), the clamp opening (CO, blue) HelD-specific domain is projected from the HelD 1A domain (yellow) towards the β′-clamp (gray). At one end, the CO is bonded to the 1A domain by the CO-linker (cyan), and stabilized by β-turn 561-563 and α19 (yellow). On the other end, the CO-domain tip abuts towards the β′-NCD three-stranded sheet. Concomitantly, the HelD helix α16 (part of peptide HelD/449–473, orange) butts against the β′/1164–1210 three-stranded sheet. The connection between α16 and the 1A-extension is disordered (dotted line). In State II (e), The CO interaction with the 1A domain remains similar to State I (d). The CO-domain tip, however, shifts towards the β′-rudder (green) and β′/122–133 α-helix. Concomitantly, the HelD PCh-loop (orange) folds towards the active site (MgA, magenta sphere) and folds back towards the 1A-extension (brick) and 1A domain. f The PCh-loop folds into the RNAP active site. The HelD loop 473–494 and the two adjacent α-helices (α16 and α17, orange) fold alongside the RNAP bridge helix (BH, cyan) towards the RNAP active site and HelD/Asp482 directly contacts the MgA (magenta sphere, details in the inset, coordination of MgA is marked with blue dotted lines). The RNAP trigger loop (TL, yellow) is restricted and folded between the HelD PCh-loop helix α17, the HelD NCC-domain (ruby), β′-BH, and the β-core domain (dark gray). g Detail of the β′-BH interaction with HelD α16 and α17. BH β′/Arg874 and Arg875 sandwich HelD/Tyr466, and β′/Tyr871 stacks on HelD/Phe502. The stacking interactions are marked with yellow dotted lines. h, i The HelD PCh-loop binding in the active site chamber is mutually exclusive with the presence of the transcription bubble. Two perpendicular views of superposition of the Tt RNAP elongation complex (PDB ID 2O5J, pale colors) and HelD State II (solid colors) are shown. The folded TL in pre-translocated EC would sterically clash with the HelD NCC-domain. The HelD PCh-loop tip would sterically clash with RNA/DNA hybrid at positions +1 to −2, and the HelD α16 and α17 helices would clash with downstream DNA duplex. Color code as in f, template DNA in pink, non-template DNA in gray, product RNA and incoming NTP at position +1 in green.