Fig. 3: An engineered proteolysis assay measures translocation by the bEBP.

a The structures suggest that the bEBP (blue; shown with C-terminal domains) actively extrudes σ^N-RI (orange) from the initiation complex (left panel). The mycobacterial proteasome requires an ATPase (Mpa, purple) to feed protein substrates (green) into its proteolytic chamber (middle panel). A short C-terminal motif (GQYL) maintains interaction between proteasome and ATPase (inset). In our assay, an engineered variant of the bEBP carrying the GQYL motif (light blue) interacts with the proteasome. Proteolysis of σ^N-RI provides a measure for bEBP-mediated translocation during transcription initiation. b Schematic representation of the variants used for the proteolytic assay. c Distinct band shifts of 2Nt-σ^N, GS7-σ^N, and Nt-σ^N in presence of ATP and the bEBP variant carrying the GQYL motif (C1-GS4) demonstrate translocation of σ^N-RI into the proteasome by the bEBP. Reactions contained 0.5 μM RNAP, 0.5 μM σ^N, 0.6 μM dhsU-CT, and 0.5 μM C1 or C1-GS4 and 5 mM ATP or ATPγS as indicated. Representative gels of at least two individual experiments are shown. d Processive translocation by the bEBP occurs with σ^N, C1-GS4, and the proteasome. A translocation halt is only observed with the full transcription complex. Representative gels of three individual experiments are shown. e Estimated length required to reach the proteasome active site predicts bEBP position on σ^N-RI during translocation halt (black shading). Secondary structure elements of σ^N-RI present in RPem (H1 and H2) are indicated as gray dashed boxes. The experimentally determined cleavage site is indicated in red (*) on the sequence of Nt-σ^N and the σ^N-AAA variant. Conserved net charge distribution of σ^N-RI (solid line) changes from positive to negative around residues 28–30. Charge distribution of σ^N-AAA (dashed line) shown for comparison. Sequence logo is based on the alignment shown in Supplementary Fig. 9. f The surface of the inner pore of the bEBP is lined with negative charges near the hydrophobic pockets that capture residues during translocation. Solvent-excluded surface was clipped to reveal the pore interior and colored according to Coulombic electrostatic potential calculated in ChimeraX. Source data are provided as a Source Data file.