RbpA relaxes promoter selectivity of M. tuberculosis RNA polymerase

Abstract

The transcriptional activator RbpA associates with Mycobacterium tuberculosis RNA polymerase (MtbRNAP) during transcription initiation, and stimulates formation of the MtbRNAP-promoter open complex (RPo). Here, we explored the influence of promoter motifs on RbpA-mediated activation of MtbRNAP containing the stress-response σB subunit. We show that both the 'extended -10' promoter motif (T-17G-16T-15G-14) and RbpA stabilized RPo and allowed promoter opening at suboptimal temperatures. Furthermore, in the presence of the T-17G-16T-15G-14 motif, RbpA was dispensable for RNA synthesis initiation, while exerting a stabilization effect on RPo. On the other hand, RbpA compensated for the lack of sequence-specific interactions of domains 3 and 4 of σB with the extended -10 and the -35 motifs, respectively. Mutations of the positively charged residues K73, K74 and R79 in RbpA basic linker (BL) had little effect on RPo formation, but affected MtbRNAP capacity for de novo transcription initiation. We propose that RbpA stimulates transcription by strengthening the non-specific interaction of the σ subunit with promoter DNA upstream of the -10 element, and by indirectly optimizing MtbRNAP interaction with initiation substrates. Consequently, RbpA renders MtbRNAP promiscuous in promoter selection, thus compensating for the weak conservation of the -35 motif in mycobacteria.

Figure 1.

RbpA is dispensable for transcription from promoters containing the extended −10 motif. (A) Schematic representation of the sigAP promoter and its derivatives. Mutated bases are underlined. (B) Representative gel of the run-off [³²P]-RNA products synthesized in the multiple-round transcription assay using sigAP-WT and the indicated derivatives in the absence and presence of RbpA. (C) Quantification of the run-off [³²P]-RNA products obtained in the transcription assay shown in panel B (mean values ± SE of three experiments except of TGTGC values which are from one experiment). All shown products were used for quantification. Values were normalized to the value obtained with the sigAP-WT promoter in the presence of RbpA. (D) KMnO₄ probing of the open complexes formed at the sigAP-WT promoter and the indicated derivatives. Promoter DNA was fluorescein-labeled on the template strand. Traces of the gel lanes are shown at the bottom (E) Number and percentage of promoters harboring the indicated extended −10 motif variants in a subset of M. tuberculosis promoters active during the exponential phase (promoters from Cortez et al. (30)). The bioinformatic analysis was performed using the UniproUGENE software (49).

Figure 2.

RbpA stabilizes open promoter complexes at the extended −10 promoter. (A) EMSA analysis of the promoter complex formation using σ^B-MtbRNAP and fluorescein-labeled sigAP promoter variants. Complexes were resolved using native 5% PAGE. (B) Quantification of the EMSA results (mean values ± SE of three experiments). (C) Effect of RbpA on sigAP-TGTG promoter binding measured by MtbRNAP titration in EMSA assays. (D) Quantification of the results shown in panel C. (E) Time-course of RPo formation monitored in a single-round run-off transcription assay. σ^B-MtbRNAP was incubated with the sigAP-WT and sigAP-TGTG promoters for the indicated times and then supplemented with NTPs and competitor poly(dI-dC). Representative gel showing the run-off [³²P]-RNA products produced during 3 min of transcription. (F) Time-course of promoter escape monitored in a single-round transcription assay. NTPs and competitor poly(dI-dC) were added to pre-formed RPo complexes and transcription was performed for the indicated times. Representative gel showing the run-off [³²P]-RNA products used for quantification. (G) Quantification of the experiments shown in E and F (mean values ± SE of three (sigAP -WT) and two (sigAP-TGTG) experiments). For each experiment, the amount of transcripts at each time-point was normalized to the plateau value. (H) The half-times of RPo formation and of promoter escape were determined from the plots shown in panel G. (I) Fluorescence fold-change during dissociation of the complexes formed by MtbRNAP at the sigAP-TGTG promoter with and without RbpA. E, MtbRNAP core enzyme (red); Eσ^B, σ^B-MtbRNAP (blue); Eσ^B + RbpA, RbpA-σ^B-MtbRNAP (black). The graph represents the average of three independent experiments. (J) Apparent kinetic and thermodynamic constants calculated from the data presented in panels D and I. t_1/2 was calculated as: t_1/2 = ln(2)/k_d.

Figure 3.

MtbRNAP forms open promoter complexes at 0°C. (A–C) Temperature-dependence of promoter melting by MtbRNAP probed with KMnO₄. DNA was labeled with fluorescein on the template strand. MtbRNAP-promoter complexes were formed at the indicated temperatures in the absence (–) or presence (+) of RbpA. (D) Quantification of the results shown in panels A and B. The open DNA fractions were calculated as the cleaved DNA to total DNA ratio.

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).

Figure 5.

Impact of σ4 and the –35 element on MtbRNAP activity. (A) Scheme showing the −35 motif of the sigAP promoter with the introduced mutations underlined. (B) Run-off [³²P]-RNA products synthesized by wild type σ^B-MtbRNAP from sigAP-WT and sigAP-TGTG and the respective variants lacking the −35 element (Δ-35). (C) Quantification of the results of the experiment shown in panel B (mean values ± SE of two experiments). (D) EMSA analysis of promoter complex formation by σ^B-MtbRNAP using the sigAP-WT and sigAP-TGTG promoters and the respective variants lacking the −35 element (Δ-35). (E) Quantification of the results shown in panel D (mean values ± SE of two experiments). (F) Analysis of the RbpA-σ^B subunit interaction by native gel electrophoresis. RbpA, labeled with DyLight 633, was incubated with increasing concentrations (0.8, 1.6, 3.2 μM) of wild type σ^B (WT) or the mutant in which domain 4 residues 252–323 were deleted (Δ4). (G) Abortive transcription activity of wild type σ^B-MtbRNAP (WT) and mutant σ^BΔ4-MtbRNAP (Δ4) on the lacUV5-bubble template harboring a heteroduplex region. (H) Run-off [³²P]-RNA products synthesized in the presence of wild type σ^B-MtbRNAP (WT) or mutant σ^BΔ4-MtbRNAP (Δ4) and the sigAP-WT, sigAP-TGTG or B. subtilis sinP3 promoter that lacks the −35 element.

Figure 6.

Effect of mutations in RbpA-BL on RPo formation and transcription initiation. (A) EMSA analysis of promoter complex formation by σ^B-MtbRNAP in the presence of the RbpA_R79A mutant. (B) EMSA analysis of promoter complex formation by σ^B-MtbRNAP in the presence of the RbpA_KKAA mutant. (C, D) Quantification of the results (mean value ± SE of two experiments) shown in panel A and B, respectively. (E) KMnO₄ probing of MtbRNAP-promoter complexes formed in the presence of the indicated RbpA mutants. (F) Run-off [³²P]-RNA products synthesized during run-off transcription assay from the sigAP promoter by σ^B-MtbRNAP in the absence or presence of the indicated RbpA variants. Transcription was performed with or without RNA primer (GpC). (G) Quantification of the results shown in panel F (mean values ± SE of two experiments).

Figure 7.

Effect of mutations in RbpA-BL on the kinetics of RPo formation and promoter escape. (A) RPo formation was monitored in single-round run-off transcription assays. (B) Promoter escape was monitored in single-round run-off transcription assays. Representative gels showing run-off [³²P]-RNA products synthesized from the sigAP-WT promoter by σ^B-MtbRNAP in the presence of the indicated RbpA variants. (C) Quantification of the experiments shown in A and B (mean values ± SE of three experiments). All shown RNA products were used for quantification. (D) The half-times of RPo formation and promoter escape were determined from the plots shown in panel C.