Figure 4.

The substitution H166A in region 3 of σ^B abolishes MtbRNAP interaction at the extended −10 motif. (A) Structural model of Mycobacterium smegmatis RNAP in complex with RbpA and promoter DNA (PDB code: 5TW1). Red ribbon, RbpA; green ribbon, σ^A subunit; gray semitransparent molecular surface, RNAP core; blue, DNA template strand; red, DNA non-template strand; orange, TG1-motif (T_-15G_-14); yellow, TG2-motif (T_-17G_-16). Residues in σ^B (H166) and RbpA (K73, K74, R79) that were mutated are shown in CPK rendering. Schematic representations of the RbpA and σ^B domains are shown at the bottom. The positions of the mutated residues are indicated. (B) Run-off [³²P]-RNA products synthesized in run-off transcription assays using sigAP promoter derivatives in the presence or not of RbpA. (C) EMSA analysis of promoter complex formation by σ^B-MtbRNAP and fluorescein-labeled sigAP promoter variants. Complexes were resolved in native 5% PAGE. (D) Quantification of the experiment shown in panel C (mean values ± SE of two experiments).