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The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase.

Tabib-Salazar A, Liu B, Doughty P, Lewis RA, Ghosh S, Parsy ML, Simpson PJ, O'Dwyer K, Matthews SJ, Paget MS - Nucleic Acids Res. (2013)

Bottom Line: RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme.Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors.The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.

ABSTRACT
RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.

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Localization of RbpASc at a σHrdB-dependent promoter in vivo. (A) Schematic of the rplJ region. The transcription start point (+1) of rplJp is indicated by a black arrow located 240 bp upstream from the rplJ start codon. The bars above the genes show the relative positions of PCR products used for ChIP–qPCR that are centred with respect to +1 as follows: 1, −653 bp; 2, −79 bp; 3, +235 bp; 4, +235 bp. (B) Occupancy of RbpASc, RNAP β subunit, and σHrdB at the indicated regions in S. coelicolor S129 (pSX190), after treatment with rifampicin. Immunoprecipitations were performed using monoclonal anti-β, polyclonal anti-σHrdB and anti-FLAG antibody to detect RbpA–FLAG. To allow comparison of RbpASc, β and σHrdB localization, after absolute quantitation of co-immunoprecipitated DNA and background correction for each antibody, enrichment is presented relative to the highest corrected signal obtained using either of the four primer pairs. Standard deviations (calculated for two biological replicates) are indicated.
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gkt277-F2: Localization of RbpASc at a σHrdB-dependent promoter in vivo. (A) Schematic of the rplJ region. The transcription start point (+1) of rplJp is indicated by a black arrow located 240 bp upstream from the rplJ start codon. The bars above the genes show the relative positions of PCR products used for ChIP–qPCR that are centred with respect to +1 as follows: 1, −653 bp; 2, −79 bp; 3, +235 bp; 4, +235 bp. (B) Occupancy of RbpASc, RNAP β subunit, and σHrdB at the indicated regions in S. coelicolor S129 (pSX190), after treatment with rifampicin. Immunoprecipitations were performed using monoclonal anti-β, polyclonal anti-σHrdB and anti-FLAG antibody to detect RbpA–FLAG. To allow comparison of RbpASc, β and σHrdB localization, after absolute quantitation of co-immunoprecipitated DNA and background correction for each antibody, enrichment is presented relative to the highest corrected signal obtained using either of the four primer pairs. Standard deviations (calculated for two biological replicates) are indicated.

Mentions: Although it is known that RbpASc is present in purified RNAP preparations, it has not yet been shown to be present in RNAP–promoter complexes in vivo. We, therefore, decided to test this using chromatin immunoprecipitation combined with quantitative PCR (ChIP–qPCR). The presence of RbpASc, σHrdB and the β subunit of RNAP was analysed at the rplJp promoter. Immunoprecipitation of β and σHrdB was performed using a commercially available monoclonal antibody (42) and a polyclonal antibody, respectively. To immunoprecipitate RbpASc, the rbpA gene was C-terminally tagged with tandem FLAG epitopes and integrated into the genome of the S. coelicolor rbpA mutant S129 at the ϕC31 attachment site using the vector pSET152. The resulting construct, pSX190, fully restored normal growth rate to S129, indicating that that the epitope tag did not impede normal RbpASc function (data not shown). S. coelicolor S129 (pSX190) was grown to mid-exponential phase before treatment with rifampicin to inhibit global RNA synthesis by trapping RNAP at promoters (43). As expected, the rplJ promoter region (qPCR product 2; Figure 2), centred −79 bp upstream from the transcription start point, was highly enriched for both σHrdB and the β subunit, with much lower enrichment seen for control regions centred −653 bp upstream, or +235 bp and +608 bp downstream, of the transcription initiation site, suggesting that σHrdB–RNAP was indeed trapped at the rplJ promoter (Figure 2). The enrichment patterns seen using the anti-FLAG antibody were similar to those obtained with the σHrdB and the β antibodies, indicating that RbpASc is present in these transcription initiation complexes, although it should be noted that differences in antibody–epitope affinities prevent the relative proportion of initiation complexes that contain RbpASc from being assessed. Control anti-FLAG immunoprecipitation experiments using an equivalent strain in which RbpASc was untagged showed no enrichment above that seen with the ‘no antibody’ control, confirming that this signal is specific to RbpASc (data not shown).Figure 2.


The actinobacterial transcription factor RbpA binds to the principal sigma subunit of RNA polymerase.

Tabib-Salazar A, Liu B, Doughty P, Lewis RA, Ghosh S, Parsy ML, Simpson PJ, O'Dwyer K, Matthews SJ, Paget MS - Nucleic Acids Res. (2013)

Localization of RbpASc at a σHrdB-dependent promoter in vivo. (A) Schematic of the rplJ region. The transcription start point (+1) of rplJp is indicated by a black arrow located 240 bp upstream from the rplJ start codon. The bars above the genes show the relative positions of PCR products used for ChIP–qPCR that are centred with respect to +1 as follows: 1, −653 bp; 2, −79 bp; 3, +235 bp; 4, +235 bp. (B) Occupancy of RbpASc, RNAP β subunit, and σHrdB at the indicated regions in S. coelicolor S129 (pSX190), after treatment with rifampicin. Immunoprecipitations were performed using monoclonal anti-β, polyclonal anti-σHrdB and anti-FLAG antibody to detect RbpA–FLAG. To allow comparison of RbpASc, β and σHrdB localization, after absolute quantitation of co-immunoprecipitated DNA and background correction for each antibody, enrichment is presented relative to the highest corrected signal obtained using either of the four primer pairs. Standard deviations (calculated for two biological replicates) are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3675491&req=5

gkt277-F2: Localization of RbpASc at a σHrdB-dependent promoter in vivo. (A) Schematic of the rplJ region. The transcription start point (+1) of rplJp is indicated by a black arrow located 240 bp upstream from the rplJ start codon. The bars above the genes show the relative positions of PCR products used for ChIP–qPCR that are centred with respect to +1 as follows: 1, −653 bp; 2, −79 bp; 3, +235 bp; 4, +235 bp. (B) Occupancy of RbpASc, RNAP β subunit, and σHrdB at the indicated regions in S. coelicolor S129 (pSX190), after treatment with rifampicin. Immunoprecipitations were performed using monoclonal anti-β, polyclonal anti-σHrdB and anti-FLAG antibody to detect RbpA–FLAG. To allow comparison of RbpASc, β and σHrdB localization, after absolute quantitation of co-immunoprecipitated DNA and background correction for each antibody, enrichment is presented relative to the highest corrected signal obtained using either of the four primer pairs. Standard deviations (calculated for two biological replicates) are indicated.
Mentions: Although it is known that RbpASc is present in purified RNAP preparations, it has not yet been shown to be present in RNAP–promoter complexes in vivo. We, therefore, decided to test this using chromatin immunoprecipitation combined with quantitative PCR (ChIP–qPCR). The presence of RbpASc, σHrdB and the β subunit of RNAP was analysed at the rplJp promoter. Immunoprecipitation of β and σHrdB was performed using a commercially available monoclonal antibody (42) and a polyclonal antibody, respectively. To immunoprecipitate RbpASc, the rbpA gene was C-terminally tagged with tandem FLAG epitopes and integrated into the genome of the S. coelicolor rbpA mutant S129 at the ϕC31 attachment site using the vector pSET152. The resulting construct, pSX190, fully restored normal growth rate to S129, indicating that that the epitope tag did not impede normal RbpASc function (data not shown). S. coelicolor S129 (pSX190) was grown to mid-exponential phase before treatment with rifampicin to inhibit global RNA synthesis by trapping RNAP at promoters (43). As expected, the rplJ promoter region (qPCR product 2; Figure 2), centred −79 bp upstream from the transcription start point, was highly enriched for both σHrdB and the β subunit, with much lower enrichment seen for control regions centred −653 bp upstream, or +235 bp and +608 bp downstream, of the transcription initiation site, suggesting that σHrdB–RNAP was indeed trapped at the rplJ promoter (Figure 2). The enrichment patterns seen using the anti-FLAG antibody were similar to those obtained with the σHrdB and the β antibodies, indicating that RbpASc is present in these transcription initiation complexes, although it should be noted that differences in antibody–epitope affinities prevent the relative proportion of initiation complexes that contain RbpASc from being assessed. Control anti-FLAG immunoprecipitation experiments using an equivalent strain in which RbpASc was untagged showed no enrichment above that seen with the ‘no antibody’ control, confirming that this signal is specific to RbpASc (data not shown).Figure 2.

Bottom Line: RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme.Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors.The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK.

ABSTRACT
RbpA is a small non-DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal σ subunit in these organisms, but not to more diverged alternative σ factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA-σ interaction is mediated by the C-terminal region of RbpA and σ domain 2, and S. coelicolor RbpA mutants that are defective in binding σ are unable to stimulate transcription in vitro and are inactive in vivo. Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor, these data support a model in which RbpA plays a key role in the σ cycle in actinobacteria.

Show MeSH
Related in: MedlinePlus