Fig. 1: Associating partners of HelD in vivo and structure of the Msm RNAP core together with σ^A, RbpA and HelD.

a Silver-stained SDS-PAGE of HelD-FLAG pull-down from exponential (EXP) and stationary (STA) phase of growth. A No-Tag strain was used as a control. Proteins pulled down with HelD are indicated on the right-hand side. The experiment was performed four times and a representative gel is shown. The dotted line shows electronic assembly of the gel. b Quantitative mass spectrometry analysis of HelD-FLAG pull-down vs No-Tag strain in EXP and STA phases of growth, respectively. The analysis was done from three biological replicates. The abundance of individual proteins was compared by two-tailed student’s t-test. The permutation-based FDR was used as an adjustment of p-value. The enrichment is shown with a volcano plot (−log₁₀ p value > 2 on the y-axis, protein enrichments > 1.5 on the x-axis). Significantly enriched proteins are shown as red (EXP) and blue (STA) dots, respectively. The identity of the most enriched proteins is indicated. c Enrichment of selected proteins from (b) (EXP and STA) related to the transcription machinery, showing relative enrichment of the proteins in the HelD-FLAG pull-down. CarD was not present in the HelD-FLAG pull-down dataset. This is indicated with the cross. Source data are provided as a Source Data file. d Color-coded annotation of Msm RNAP core, domains of HelD, σ^A and RbpA. e, f Two conformations of the Msm RNAP core complex together with σ^A, RbpA and HelD in state I (HelD-holo-I) and state II (HelD-holo-II), respectively. Individual domains are color-coded according to (d). g Magnified details of panel (f). The mutual interaction of σ^A and HelD in the context of the β′-clamp. σ^A₂ interacts with the conserved binding site on the β′-clamp coiled-coil domain (β′-clamp CC, gray) near the β′-clamp rudder (green). The σ^A_N-helix and adjacent regions (red) wrap with specific protein–protein interactions around the HelD–CO-tip helix-turn-helix (HTH) motif (light blue). The HelD–CO-tip is also buttressed by helices α1 and α4 of the σ^A₂ domain (purple). h Magnified details of panel (g). Specific residues important for the σ^A–HelD interaction are highlighted. σ^A/Phe140 (red) and its interaction with HelD defines the beginning of ordered regions of σ^A.