Mycobacterium tuberculosis RbpA protein is a new type of transcriptional activator that stabilizes the σ A-containing RNA polymerase holoenzyme

Abstract

RbpA is an RNA polymerase (RNAP)-binding protein whose presence increases the tolerance levels of Mycobacteria to the first-line anti-tuberculosis drug rifampicin by an unknown mechanism. Here, we show that the role of Mycobacterium tuberculosis RbpA in resistance is indirect because it does not affect the sensitivity of RNAP to rifampicin while it stimulates transcription controlled by the housekeeping σ(A)-factor. The transcription regulated by the stress-related σ(F) was not affected by RbpA. The binding site of RbpA maps to the RNAP β subunit Sandwich-Barrel Hybrid Motif, which has not previously been described as an activator target and does not overlap the rifampicin binding site. Our data suggest that RbpA modifies the structure of the core RNAP, increases its affinity for σ(A) and facilitates the assembly of the transcriptionally competent promoter complexes. We propose that RbpA is an essential partner which advantages σ(A) competitiveness for core RNAP binding with respect to the alternative σ factors. The RbpA-driven stimulation of the housekeeping gene expression may help Mycobacteria to tolerate high rifampicin levels and to adapt to the stress conditions during infection.

Figure 1.

Effects of RbpA on σ^A and σ^F dependent transcription. (A and B): Sequences of the rrnAP3 and usfXP1 promoters. The −10 and −35 consensus elements are underlined, and transcription start sites (TSS) are marked by arrows. (C and D) Abortive transcription carried out by the σ^A-containing RNAP (Eσ^A) on the rrnAP3 template and σ^F-containing RNAP (Eσ^F) on the usfXP1 template. Lanes 2–5: RbpA was added to 0.15, 0.3, 0.6 and 1.2 µM, respectively. Quantification of the bands indicated by arrows is shown on the bottom graphs. The RNA amounts were normalized to the value obtained without RbpA (lane 1). The error bars are the SD of triplicate experiments. (E and F) Kinetics of single-round run-off transcription carried out by σ^A-containing RNAP on the rrnAP3 and by σ^F-containing RNAP on the usfXP1 template, respectively. RbpA (1.2 μM) was added to the reaction when indicated. The quantification of the run-off RNA is shown on the bottom graphs. The RNA amounts were normalized to the amount of RNA synthesized without RbpA after 10 min of transcription (rrnAP3) or 5 min of transcription (usfXP1). The error bars are the SD of duplicate experiments.

Figure 2.

RbpA stimulates the formation of the stable RNAP–promoter complexes. (A) EMSA of the σ^A-containing RNAP (Eσ^A) and the rrnAP3 promoter fragment. RbpA at 1.2 μM, GTP at 1 mM and heparin at 10 µg/ml were added when indicated. The promoter fragments were end-labeled by fluorescein. (B) Quantification of the experiment shown in (A). The bar graph shows the fluorescence intensity of promoter DNA bound to RNAP. (C) EMSA of the complexes of the σ^A-containing RNAP and the rrnAP3 promoter fragments formed in the absence or in the presence of 0.15, 0.3, 0.6 or 1.2 µM RbpA. (D) EMSA of the complexes of the σ^A-containing RNAP and the sinP3 promoter fragment formed in the absence or in presence of 75, 150, 300 or 600 nM RbpA. (E) EMSA of the σ^F-containing RNAP and the usfXP1 promoter fragment in the absence or in the presence of 0.15, 0.3, 0.6 or 1.2 µM of RbpA. The promoter complexes shown in (C–E) were challenged with poly(dA–dT). The RNAP–promoter complex is indicated as ‘RP’, and non-bound DNA is indicated as ‘Free DNA’. (F) Quantification of the experiments shown in (C–E).

Figure 3.

RbpA stabilizes σ^A-core RNAP interactions. (A and B) FLAG-tag pull-down experiments were performed using the RNAP core enzyme containing a FLAG-tagged β subunit. RNAP was incubated with the 6×His-tagged σ^A (A) or σ^F (B) in the presence or absence of 6×His-tagged RbpA and captured by anti-FLAG agarose. After washing, the proteins retained by the resin (lanes 3 and 4) and the total proteins in the input (lanes 1,2) were analyzed by western blotting using anti-FLAG (β panel), anti-polyhistidine (σ panel) and anti-C-term 6×His (RbpA/ω panel) antibodies. (C and D) Abortive transcription initiated by core RNAP (300 nM) and increasing concentrations (37.5, 75, 150, 300, 600, 900, 1800 and 3600 nM) of σ^A (C) and σ^F (D) at the rrnAP3 and usfXP1 promoters, respectively. RbpA was added to 2.4 µM when indicated. The [³²P]-labeled abortive RNA products are shown in the top panels. The graphs show normalized amounts of abortive RNA products synthesized at the indicated concentrations of the σ subunit. Mean values and SD of two independent experiments are shown.

Figure 4.

Cleavage of the β subunit of the RNAP core and holoenzyme by FeBABE–RbpA. Western blot analysis of the β subunit fragments stained either at the N-terminus by the anti-FLAG antibodies (A) or at the C-terminus by the anti-HA antibodies (B). FeBABE–RbpA cleavage reactions were performed either with core RNAP (marked ‘C’, lanes 2–4) or holoenzyme (marked ‘H’, lane 5). Control cleavage reactions were performed either without FeBABE and RbpA (lane 2) or with unmodified RbpA (lane 3). The protein ladder was generated by the cleavage of the β subunit at Cys residues using Cys-specific cleavage reagent, TNCBA (lane 6). Numbering along the right edge of the gels shows the positions of the Cys-specific cleavages on the β subunit. A commercial molecular weight marker is shown in lane 1 of each gel. (C) A diagram showing the β subunit with the FLAG-tag shown as an N-terminal yellow box and the HA-tag as a C-terminal green box. The gray shaded areas correspond to the evolutionarily conserved regions A–I. The red shaded areas mark the clusters of rifampicin resistance (3,49). NTCBA-cleaved Cys residues are indicated by black triangles. The positions of the FeBABE–RbpA cleavage sites calculated from the experiments presented in (A and B) are shown as cyan rectangles. (D) A model of the FeBABE–RbpA cleavage sites on the structure of T. thermophilus RNAP holoenzyme in complex with DNA (32,33). DNA is shown as a ladder model with the template strand in red and the non-template strand in blue. The β (gray), β′ (pink), α (yellow) subunits are shown as molecular surface models. The σ subunit (green) is shown as a ribbon model. The RbpA-binding regions are colored in cyan. The surface of the rifampicin-resistance cluster I (RIF cluster I), spanning the M. tuberculosis β residues 425–454, is colored in orange. Molecular graphics images were produced using the UCSF Chimera package (50).

Figure 5.

Influence of RbpA on RNAP inhibition by rifampicin. (A) Abortive transcription initiation from the rrnAP3 promoter by the σ^A-containing RNAP in the presence of [³²P]-UTP, ATP and GTP. Rifampicin (Rif) was added to the reactions at the indicated concentrations. RbpA (1.2 μM) was added to the reactions when indicated. (B) Quantification of the experiment shown in (A). The amounts of abortive RNA synthesized in the presence of different concentrations of rifampicin were normalized to the amount of the RNA synthesized without rifampicin (the first lane) and plotted against rifampicin concentrations. The error bars are the SD of duplicate experiments.