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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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Related in: MedlinePlus

RbpASc binds to the σ2 domain of σHrdB. (A) A schematic diagram indicating the four conserved regions/globular domains of σHrdB together with BACTH interaction data between truncated T25–σHrdB fusions and RbpASc–T18. The T25–σHrdB fusions included the following amino acids: σ1.1–σ2, 1–347; σ2, 211–347; σ3–σ4, 348–511; σ2–σ4, 211–511; σ4, 435–511. Control strains with pKT25 and pUT18–rbpA exhibited <1% the activity of the σ2–σ4 interaction. Experiments were performed in triplicate, and standard deviations are indicated. (B) Interaction between His6-tagged σHrdB fragments and RbpASc as judged using in vitro pull-down experiments. Purified His6-tagged σHrdB fragments (closed diamond) were mixed with RbpASc (∼0.5–1 μM each) before purification using Ni-affinity magnetic beads. Eluted proteins were separated by 4–12% Bis–Tris SDS–PAGE and stained using Coomassie brilliant blue. RbpASc is indicated with a black arrowhead. Closed square, unknown contaminating protein.
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gkt277-F7: RbpASc binds to the σ2 domain of σHrdB. (A) A schematic diagram indicating the four conserved regions/globular domains of σHrdB together with BACTH interaction data between truncated T25–σHrdB fusions and RbpASc–T18. The T25–σHrdB fusions included the following amino acids: σ1.1–σ2, 1–347; σ2, 211–347; σ3–σ4, 348–511; σ2–σ4, 211–511; σ4, 435–511. Control strains with pKT25 and pUT18–rbpA exhibited <1% the activity of the σ2–σ4 interaction. Experiments were performed in triplicate, and standard deviations are indicated. (B) Interaction between His6-tagged σHrdB fragments and RbpASc as judged using in vitro pull-down experiments. Purified His6-tagged σHrdB fragments (closed diamond) were mixed with RbpASc (∼0.5–1 μM each) before purification using Ni-affinity magnetic beads. Eluted proteins were separated by 4–12% Bis–Tris SDS–PAGE and stained using Coomassie brilliant blue. RbpASc is indicated with a black arrowhead. Closed square, unknown contaminating protein.

Mentions: The different domains of σ have distinct functions; therefore, to better understand the role of RbpA in transcription initiation, we sought to localize the interaction to individual domain(s) of σHrdB. The σ2, σ3 and σ4 structural domains of σHrdB were predicted based on the structures of σA from Thermus sp. (11,51), and five overlapping T25–hrdB fusions were constructed and tested for interaction with the rbpA–T18 fusion in BACTH analysis (Figure 7). No interaction was detected for T25–hrdB (σ3–σ4) or T25–hrdB (σ4). However, all fragments that included σ2 interacted, including T25–hrdB (σ2) (amino acid residues 211–347), which comprises conserved regions 1.2–2.4. To confirm these data, each of the hrdB constructs used in the BACTH analysis was overexpressed with an N-terminal His6-tag, and the corresponding proteins purified, then tested for interaction with RbpASc using an in vitro pull-down assay (apart from σ1.1–σ2, which was insoluble). The pure σHrdB fragments (domains σ2, σ2–σ4, σ3–σ4 and σ4) were mixed with native RbpASc for 15 min and then isolated using magnetic Ni-affinity beads. RbpASc was co-isolated with the σ2 and σ2–σ4 fragments of σHrdB but not with σ3–σ4 and σ4, confirming a direct and specific interaction with the σ2 domain. Using both BACTH and in vitro pull downs, we also detected equivalent interactions between the σ2 domain of σA and the RbpAMt (data not shown). Furthermore, when equivalent σ2 and RbpA proteins from S. coelicolor and M. tuberculosis are co-expressed in E. coli, with one partner His6-tagged, they co-elute as a complex during Ni-affinity chromatography (data not shown). Taken together, these data suggest that the σ2–RbpA interaction involving the principal σ factor is conserved across the actinobacteria.Figure 7.


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)

RbpASc binds to the σ2 domain of σHrdB. (A) A schematic diagram indicating the four conserved regions/globular domains of σHrdB together with BACTH interaction data between truncated T25–σHrdB fusions and RbpASc–T18. The T25–σHrdB fusions included the following amino acids: σ1.1–σ2, 1–347; σ2, 211–347; σ3–σ4, 348–511; σ2–σ4, 211–511; σ4, 435–511. Control strains with pKT25 and pUT18–rbpA exhibited <1% the activity of the σ2–σ4 interaction. Experiments were performed in triplicate, and standard deviations are indicated. (B) Interaction between His6-tagged σHrdB fragments and RbpASc as judged using in vitro pull-down experiments. Purified His6-tagged σHrdB fragments (closed diamond) were mixed with RbpASc (∼0.5–1 μM each) before purification using Ni-affinity magnetic beads. Eluted proteins were separated by 4–12% Bis–Tris SDS–PAGE and stained using Coomassie brilliant blue. RbpASc is indicated with a black arrowhead. Closed square, unknown contaminating protein.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt277-F7: RbpASc binds to the σ2 domain of σHrdB. (A) A schematic diagram indicating the four conserved regions/globular domains of σHrdB together with BACTH interaction data between truncated T25–σHrdB fusions and RbpASc–T18. The T25–σHrdB fusions included the following amino acids: σ1.1–σ2, 1–347; σ2, 211–347; σ3–σ4, 348–511; σ2–σ4, 211–511; σ4, 435–511. Control strains with pKT25 and pUT18–rbpA exhibited <1% the activity of the σ2–σ4 interaction. Experiments were performed in triplicate, and standard deviations are indicated. (B) Interaction between His6-tagged σHrdB fragments and RbpASc as judged using in vitro pull-down experiments. Purified His6-tagged σHrdB fragments (closed diamond) were mixed with RbpASc (∼0.5–1 μM each) before purification using Ni-affinity magnetic beads. Eluted proteins were separated by 4–12% Bis–Tris SDS–PAGE and stained using Coomassie brilliant blue. RbpASc is indicated with a black arrowhead. Closed square, unknown contaminating protein.
Mentions: The different domains of σ have distinct functions; therefore, to better understand the role of RbpA in transcription initiation, we sought to localize the interaction to individual domain(s) of σHrdB. The σ2, σ3 and σ4 structural domains of σHrdB were predicted based on the structures of σA from Thermus sp. (11,51), and five overlapping T25–hrdB fusions were constructed and tested for interaction with the rbpA–T18 fusion in BACTH analysis (Figure 7). No interaction was detected for T25–hrdB (σ3–σ4) or T25–hrdB (σ4). However, all fragments that included σ2 interacted, including T25–hrdB (σ2) (amino acid residues 211–347), which comprises conserved regions 1.2–2.4. To confirm these data, each of the hrdB constructs used in the BACTH analysis was overexpressed with an N-terminal His6-tag, and the corresponding proteins purified, then tested for interaction with RbpASc using an in vitro pull-down assay (apart from σ1.1–σ2, which was insoluble). The pure σHrdB fragments (domains σ2, σ2–σ4, σ3–σ4 and σ4) were mixed with native RbpASc for 15 min and then isolated using magnetic Ni-affinity beads. RbpASc was co-isolated with the σ2 and σ2–σ4 fragments of σHrdB but not with σ3–σ4 and σ4, confirming a direct and specific interaction with the σ2 domain. Using both BACTH and in vitro pull downs, we also detected equivalent interactions between the σ2 domain of σA and the RbpAMt (data not shown). Furthermore, when equivalent σ2 and RbpA proteins from S. coelicolor and M. tuberculosis are co-expressed in E. coli, with one partner His6-tagged, they co-elute as a complex during Ni-affinity chromatography (data not shown). Taken together, these data suggest that the σ2–RbpA interaction involving the principal σ factor is conserved across the actinobacteria.Figure 7.

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