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Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes.

Blomberg J, Aguilar X, Brännström K, Rautio L, Olofsson A, Wittung-Stafshede P, Björklund S - Nucleic Acids Res. (2012)

Bottom Line: Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes.However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25.We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.

ABSTRACT
Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

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Related in: MedlinePlus

Structural similarity between the ACID-domains of Med25 from human and A. thaliana mediates similar binding propensity to Dreb2af.l.. (A) Prediction of secondary structure in the ACID-domain of Med25 from human (humMed25394–543) and A. thaliana (aMed25551–680) was made utilizing the jpred 3 server. The humMed25 contains somewhat more α-helixes (H) in dark grey and β-strands (E) in light grey than aMed25. (B) CD spectra of humMed25394–543 and aMed25551–680 confirmed that humMed25394–543 contains more ordered structures. (C) Dreb2af.l. (D) was bound to IgG sepharose through its GB-1 tag. Addition of humMed25394–543 (hM) resulted in a Dreb2af.l.–humMed25394–543 complex that was visualized by SDS–PAGE and immunoblotting. (D) Complex formation of Dreb2af.l. and humMed25394–543 (hM) was investigated with formaldehyde cross-linking (0.5%) and immunoblot. Dreb2af.l., and humMed25394–543 was mixed at 1:1 (1 µM each) and 1:4 (1 µM Dreb2af.l. and 4 µM Med25) ratios. An amount of 4 µM humMed25394–543 alone, and Dreb2af.l. incubation with GST (1:4) severed as negative controls. Complex formation between 1 µM aMed25551–680 (M) and 1 µM Dreb2af.l. was included as a positive control. (E) SRP sensograms showing the binding of increasing concentrations of humMed25394–543 to Df.l.. An amount of 3 µM of humMed25394–543 was diluted 1:1 down to 0.047 µM and samples were passed over a NTA sensor chip sensorchip where Df.l. was immobilized via its His-tag.
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gks265-F7: Structural similarity between the ACID-domains of Med25 from human and A. thaliana mediates similar binding propensity to Dreb2af.l.. (A) Prediction of secondary structure in the ACID-domain of Med25 from human (humMed25394–543) and A. thaliana (aMed25551–680) was made utilizing the jpred 3 server. The humMed25 contains somewhat more α-helixes (H) in dark grey and β-strands (E) in light grey than aMed25. (B) CD spectra of humMed25394–543 and aMed25551–680 confirmed that humMed25394–543 contains more ordered structures. (C) Dreb2af.l. (D) was bound to IgG sepharose through its GB-1 tag. Addition of humMed25394–543 (hM) resulted in a Dreb2af.l.–humMed25394–543 complex that was visualized by SDS–PAGE and immunoblotting. (D) Complex formation of Dreb2af.l. and humMed25394–543 (hM) was investigated with formaldehyde cross-linking (0.5%) and immunoblot. Dreb2af.l., and humMed25394–543 was mixed at 1:1 (1 µM each) and 1:4 (1 µM Dreb2af.l. and 4 µM Med25) ratios. An amount of 4 µM humMed25394–543 alone, and Dreb2af.l. incubation with GST (1:4) severed as negative controls. Complex formation between 1 µM aMed25551–680 (M) and 1 µM Dreb2af.l. was included as a positive control. (E) SRP sensograms showing the binding of increasing concentrations of humMed25394–543 to Df.l.. An amount of 3 µM of humMed25394–543 was diluted 1:1 down to 0.047 µM and samples were passed over a NTA sensor chip sensorchip where Df.l. was immobilized via its His-tag.

Mentions: Purification of Med25551–680 and Dreb2a168–335 was carried out as described previously (16). The GST-tag of Med25551–680 that is removed by cleavage with Prescission Enzyme (GE Healthcare) was collected and stored in −20°C before use as a control for the experiments described in Figure 7. The nucleotide sequence corresponding to amino acids 168–253 (Dreb2a168–253) and 254–335 (Dreb2a254–335) was amplified from cDNA with polymerase chain reaction and cloned into the NcoI/NotI sites of the pETM-6×his vector (kindly provided by Günter Stier, EMBL, Germany). The primers used were: 5′-GCTACCATGGATTGTGAATCTAAACCCTTCT-3′and 5′-GCTTGCGGCCGCTTACAAGTGACTCTGATCCACATG-3′ for Dreb2a168–253 and 5′-GCTACCATGGATTCTTCAGACATGTTTGATG-3′ and 5′-GCTTGCGGCCGCTTAGTTCTCCAGATCCAAGTAACT-3′ for Dreb2a254–335. Dreb2a168–253 and Dreb2a245–335 were cloned into the pETM-6×his vector and produced as described for Dreb2a168–335 (16). Final purification of Dreb2a168–253 and Dreb2a254–335 was made with ion exchange chromatography using a Mono Q 5/50 GL (GE Healthcare). The proteins were applied to the columns in 25 mM sodium phosphate buffer pH 6.2 and sodium acetate buffer pH 5.2, respectively. Both buffers contained 1 mM dithiothreitol (DTT) and a starting concentration of 20 mM NaCl. Adsorbed proteins were eluted with a linear gradient of 18 column volumes to 0.8 M NaCl on an ÄKTAexplorer (GE Healthcare). Full-length Dreb2a, Dreb2af.1., was cloned into a pETM-6×his-GB1 vector at the NcoI/NotI sites and the resulting plasmid was transfected into the Escherichia coli Rosetta (DE3) pLysS strain. The bacteria were grown at 30°C in Terrific Broth supplemented with 100 µg/ml kanamycin and 34 µg/ml chloramphenicol. Protein expression was induced by addition of isopropyl β-d-thiogalactopyranoside (IPTG) to 1 mM at OD600 = 1.5. After 6 h of induction, cells were harvested by centrifugation and pellets were lysed in 20 mM Tris pH 8.0, 300 mM NaCl, 0.2% NP40 and 2 mM β-mercaptoethanol. The lysates were sonicated on ice with a Branson Sonifier 450 equipped with a microtip (VWR, Sweden). The following settings were applied: duty cycle 50%, output control 3.5, time 8 min. The lysates were then cleared by centrifugation at 35 000 g for 1 h using a 45Ti rotor (Beckman Coulter AB, Sweden), and Dreb2af.1. was purified from the supernatant by ammonium sulfate precipitation (20%). Precipitated proteins were dissolved in 20 mM Tris pH 7.5, 100 mM NaCl and 2 mM β-mercaptoethanol and loaded onto a Mono S 5/50 GL (GE Healthcare). Adsorbed proteins were eluted with a linear gradient to 800 mM NaCl. Fractions containing Dreb2af.1. were pooled, and dialyzed against 20 mM Tris pH 7.5, 150 mM NaCl and 2 mM β-mercaptoethanol.


Interactions between DNA, transcriptional regulator Dreb2a and the Med25 mediator subunit from Arabidopsis thaliana involve conformational changes.

Blomberg J, Aguilar X, Brännström K, Rautio L, Olofsson A, Wittung-Stafshede P, Björklund S - Nucleic Acids Res. (2012)

Structural similarity between the ACID-domains of Med25 from human and A. thaliana mediates similar binding propensity to Dreb2af.l.. (A) Prediction of secondary structure in the ACID-domain of Med25 from human (humMed25394–543) and A. thaliana (aMed25551–680) was made utilizing the jpred 3 server. The humMed25 contains somewhat more α-helixes (H) in dark grey and β-strands (E) in light grey than aMed25. (B) CD spectra of humMed25394–543 and aMed25551–680 confirmed that humMed25394–543 contains more ordered structures. (C) Dreb2af.l. (D) was bound to IgG sepharose through its GB-1 tag. Addition of humMed25394–543 (hM) resulted in a Dreb2af.l.–humMed25394–543 complex that was visualized by SDS–PAGE and immunoblotting. (D) Complex formation of Dreb2af.l. and humMed25394–543 (hM) was investigated with formaldehyde cross-linking (0.5%) and immunoblot. Dreb2af.l., and humMed25394–543 was mixed at 1:1 (1 µM each) and 1:4 (1 µM Dreb2af.l. and 4 µM Med25) ratios. An amount of 4 µM humMed25394–543 alone, and Dreb2af.l. incubation with GST (1:4) severed as negative controls. Complex formation between 1 µM aMed25551–680 (M) and 1 µM Dreb2af.l. was included as a positive control. (E) SRP sensograms showing the binding of increasing concentrations of humMed25394–543 to Df.l.. An amount of 3 µM of humMed25394–543 was diluted 1:1 down to 0.047 µM and samples were passed over a NTA sensor chip sensorchip where Df.l. was immobilized via its His-tag.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401450&req=5

gks265-F7: Structural similarity between the ACID-domains of Med25 from human and A. thaliana mediates similar binding propensity to Dreb2af.l.. (A) Prediction of secondary structure in the ACID-domain of Med25 from human (humMed25394–543) and A. thaliana (aMed25551–680) was made utilizing the jpred 3 server. The humMed25 contains somewhat more α-helixes (H) in dark grey and β-strands (E) in light grey than aMed25. (B) CD spectra of humMed25394–543 and aMed25551–680 confirmed that humMed25394–543 contains more ordered structures. (C) Dreb2af.l. (D) was bound to IgG sepharose through its GB-1 tag. Addition of humMed25394–543 (hM) resulted in a Dreb2af.l.–humMed25394–543 complex that was visualized by SDS–PAGE and immunoblotting. (D) Complex formation of Dreb2af.l. and humMed25394–543 (hM) was investigated with formaldehyde cross-linking (0.5%) and immunoblot. Dreb2af.l., and humMed25394–543 was mixed at 1:1 (1 µM each) and 1:4 (1 µM Dreb2af.l. and 4 µM Med25) ratios. An amount of 4 µM humMed25394–543 alone, and Dreb2af.l. incubation with GST (1:4) severed as negative controls. Complex formation between 1 µM aMed25551–680 (M) and 1 µM Dreb2af.l. was included as a positive control. (E) SRP sensograms showing the binding of increasing concentrations of humMed25394–543 to Df.l.. An amount of 3 µM of humMed25394–543 was diluted 1:1 down to 0.047 µM and samples were passed over a NTA sensor chip sensorchip where Df.l. was immobilized via its His-tag.
Mentions: Purification of Med25551–680 and Dreb2a168–335 was carried out as described previously (16). The GST-tag of Med25551–680 that is removed by cleavage with Prescission Enzyme (GE Healthcare) was collected and stored in −20°C before use as a control for the experiments described in Figure 7. The nucleotide sequence corresponding to amino acids 168–253 (Dreb2a168–253) and 254–335 (Dreb2a254–335) was amplified from cDNA with polymerase chain reaction and cloned into the NcoI/NotI sites of the pETM-6×his vector (kindly provided by Günter Stier, EMBL, Germany). The primers used were: 5′-GCTACCATGGATTGTGAATCTAAACCCTTCT-3′and 5′-GCTTGCGGCCGCTTACAAGTGACTCTGATCCACATG-3′ for Dreb2a168–253 and 5′-GCTACCATGGATTCTTCAGACATGTTTGATG-3′ and 5′-GCTTGCGGCCGCTTAGTTCTCCAGATCCAAGTAACT-3′ for Dreb2a254–335. Dreb2a168–253 and Dreb2a245–335 were cloned into the pETM-6×his vector and produced as described for Dreb2a168–335 (16). Final purification of Dreb2a168–253 and Dreb2a254–335 was made with ion exchange chromatography using a Mono Q 5/50 GL (GE Healthcare). The proteins were applied to the columns in 25 mM sodium phosphate buffer pH 6.2 and sodium acetate buffer pH 5.2, respectively. Both buffers contained 1 mM dithiothreitol (DTT) and a starting concentration of 20 mM NaCl. Adsorbed proteins were eluted with a linear gradient of 18 column volumes to 0.8 M NaCl on an ÄKTAexplorer (GE Healthcare). Full-length Dreb2a, Dreb2af.1., was cloned into a pETM-6×his-GB1 vector at the NcoI/NotI sites and the resulting plasmid was transfected into the Escherichia coli Rosetta (DE3) pLysS strain. The bacteria were grown at 30°C in Terrific Broth supplemented with 100 µg/ml kanamycin and 34 µg/ml chloramphenicol. Protein expression was induced by addition of isopropyl β-d-thiogalactopyranoside (IPTG) to 1 mM at OD600 = 1.5. After 6 h of induction, cells were harvested by centrifugation and pellets were lysed in 20 mM Tris pH 8.0, 300 mM NaCl, 0.2% NP40 and 2 mM β-mercaptoethanol. The lysates were sonicated on ice with a Branson Sonifier 450 equipped with a microtip (VWR, Sweden). The following settings were applied: duty cycle 50%, output control 3.5, time 8 min. The lysates were then cleared by centrifugation at 35 000 g for 1 h using a 45Ti rotor (Beckman Coulter AB, Sweden), and Dreb2af.1. was purified from the supernatant by ammonium sulfate precipitation (20%). Precipitated proteins were dissolved in 20 mM Tris pH 7.5, 100 mM NaCl and 2 mM β-mercaptoethanol and loaded onto a Mono S 5/50 GL (GE Healthcare). Adsorbed proteins were eluted with a linear gradient to 800 mM NaCl. Fractions containing Dreb2af.1. were pooled, and dialyzed against 20 mM Tris pH 7.5, 150 mM NaCl and 2 mM β-mercaptoethanol.

Bottom Line: Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes.However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25.We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.

ABSTRACT
Mediator is a multiprotein coregulatory complex that conveys signals from DNA-bound transcriptional regulators to the RNA polymerase II transcription machinery in eukaryotes. The molecular mechanisms for how these signals are transmitted are still elusive. By using purified transcription factor Dreb2a, mediator subunit Med25 from Arabidopsis thaliana, and a combination of biochemical and biophysical methods, we show that binding of Dreb2a to its canonical DNA sequence leads to an increase in secondary structure of the transcription factor. Similarly, interaction between the Dreb2a and Med25 in the absence of DNA results in conformational changes. However, the presence of the canonical Dreb2a DNA-binding site reduces the affinity between Dreb2a and Med25. We conclude that transcription regulation is facilitated by small but distinct changes in energetic and structural parameters of the involved proteins.

Show MeSH
Related in: MedlinePlus