An activator binding module of yeast RNA polymerase II holoenzyme.
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):2967-76.An activator binding module of yeast RNA polymerase II holoenzyme.1, , , , .1Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University College of Medicine, Kangnam-ku, Seoul 135-230, Korea.AbstractThe Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system. This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter. Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays. In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme. These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity.PMID:
[PubMed - indexed for MEDLINE] PMCID: PMC84091 Subunit compositions of mutant holoenzymes. (A) Immunoprecipitation analysis. Holoenzymes (MonoQ fractions) were prepared from wild-type (YCL10; W), rgr1Δ2 (DY2010; R), gal11Δ (HS301; G), hrs1Δ (SSAB-2CF; H), and srb5Δ (CTY153; S) strains and immunoprecipitated with anti-Rgr1 antibody–beads as described in Materials and Methods. Proteins were resolved on an SDS–10% polyacrylamide gel and visualized by silver staining. The positions of core polymerase subunits (Rpb) and Mediator components are indicated at the left and right, respectively. (B) Immunoblot analysis. Wild-type (W) and mutant (R, rgr1Δ2; S, srb5Δ; G, gal11Δ; H, hrs1Δ) holoenzymes (MonoQ fractions) were subjected to immunoblot analysis with antisera specific for the PolII and Mediator components indicated between the panels. Mol Cell Biol. ):.In vitro transcription of mutant holoenzymes. Wild-type and mutant holoenzymes (700 ng each) containing the same levels of nonspecific transcriptional elongation activities were analyzed for their promoter-specific transcriptional activities in the presence of the indicated activators in an in vitro transcription system reconstituted with pure general transcription factors and other supplements as described previously (21). RNA transcripts from templates containing either the Gal4 binding site (GAL4:G-) or the GCN4 binding site (GCN4:G-) are indicated. (A) Transcriptional defects of rgr1Δ2 and srb5Δ holoenzymes. The specifically initiated transcripts from reactions that contained wild-type (W), rgr1Δ2 (R), and srb5Δ (S) holoenzymes in the absence (none) or presence of activator protein (Gal4VP16 or GCN4; 30 ng each) are shown. (B) Transcriptional defects of gal11Δ and hrs1Δ holoenzymes. The specifically initiated transcripts from reactions that contained wild-type, gal11Δ, and hrs1Δ holoenzymes in the absence (-) or presence of activator protein (Gal4VP16 [V] or GCN4 [N]; 30 ng each) are shown.Mol Cell Biol. ):.TFIIH-dependent CTD phosphorylation of mutant holoenzymes. (A) Core polymerase (core-polII, 0.3 μg) and holoenzyme (Holo-polII, 0.9 μg) were incubated with [γ-32P]ATP and without (-) or with (+) the indicated supplements (Gal4VP16 [Gal4VP, 30 ng], TBP [50 ng], TFIIB [50 ng], TFIIE [60 ng], TFIIH [60 ng], TFIIF [F, 20 ng], and DNA [pJJ470, 200 ng]) in transcription buffer for 10 min (holoenzyme) or 40 min (core polymerase) at 25°C. Phosphorylated Rpb1 was analyzed by SDS-PAGE (7.5% gel) and visualized by autoradiography. (B) CTD phosphorylation of mutant holoenzymes during Gal4VP16-mediated transcriptional activation. The degrees of CTD phosphorylation of the indicated holoenzymes (900 ng) under various transcription reaction conditions are shown. CTD phosphorylation by TFIIH (60 ng) only in the presence of the DNA template (pJJ470, 200 lane H) and under basal (lane B) or activated (Gal4VP16 mediated, lane A) transcription conditions is shown. (C) CTD phosphorylation of mutant holoenzymes during GCN4-mediated transcriptional activation. CTD of wild-type (W), rgr1Δ2 (R), srb5Δ (S), gal11Δ (G), and hrs1Δ (H) holoenzymes was phosphorylated by TFIIH under basal or activated (GCN4-mediated) transcription conditions.Mol Cell Biol. ):.Interactions of transcriptional activators with wild-type and mutant holoenzymes. (A, B, and D) Plotted is the change in the refractory index units (RU) versus time after injection of the indicated holoenzymes onto an activator-immobilized Biosensor chip. (A) SPR analysis of the interactions between Gal4VP16 and wild-type and rgr1Δ2 holoenzymes (70 μl of a MonoQ fraction at a concentration of 200 μg/ml). (B) SPR analysis of the interactions between wild-type (W) and hrs1Δ (H) holoenzymes (50 μl of a MonoQ fraction at a concentration of 250 μg/ml) and GST-VP16 or GCN4. (C) Coimmunoprecipitation (I.P.) of Gal4VP16 with wild-type (W) and mutant (R, rgr1Δ2) holoenzymes followed by Western blotting. Each holoenzyme (MonoQ fraction, 10 μg) was mixed with Gal4VP16 (300 ng) and immunoprecipitated with anti-Rgr1 antibody–beads (see Materials and Methods). In order to measure the amounts of precipitated Gal4VP16 and holoenzymes, we included equimolar amounts of recombinant Gal4VP16 and Med6 proteins (r-P; Med6, 80 Gal4VP16, 60 ng) as quantitative standards. Immunoblot analyses with antisera specific for the proteins indicated to the right of the panel are shown. (D) SPR analysis of the interactions between wild-type (W, 150 μg/ml) and srb5Δ (S, 450 μg/ml) holoenzymes and an activator (GST-VP16). Due to the low specific concentration of the srb5Δ holoenzyme in the MonoQ fraction (one-fifth the wild-type level), a threefold-larger amount of srb5Δ MonoQ fraction was injected to supply an amount of the srb5Δ holoenzyme comparable to that of the wild-type enzyme.Mol Cell Biol. ):.Direct interaction of Gal11 with acidic activators. (A) Far-Western analysis of holoenzymes with Gal4VP16. Wild-type (W) and gal11Δ (G) holoenzymes (immunopurified from a MonoQ fraction with an anti-Rgr1 antibody column, ~2 μg each) were resolved on an SDS–10% polyacrylamide gel and transferred to a nitrocellulose membrane. The proteins were renatured and allowed to bind to 32P-labeled Gal4VP16 as described in Materials and Methods. The position of the Gal11 protein was revealed by Western analysis of the same blot with the use of anti-Gal11 antiserum (αGal11). (B) Column binding assay for interactions between Gal11 and acidic activators. Agarose beads conjugated with purified GST or GST-Gal11 (5 μg each) were incubated overnight at 4°C with 10 μl of in vitro-translated, 35S-labeled Gal4VP16, Gal4VP16Δ456FP442, or GCN4. After extensive washing, the beads were boiled in SDS sample buffer, and the proteins were resolved by SDS–12.5% PAGE and subjected to autoradiography. Load represents 10% of the amount of in vitro-translated products loaded on the beads.Mol Cell Biol. ):.Simultaneous interactions of an activator with a holoenzyme and TBP. The ΔRU during the time course of the SPR analysis with a TBP-immobilized Biosensor chip is shown. Gal4VP16 (30 μg/ml, 30 μl) and wild-type holoenzyme (Holo-polII; 300 μg/ml, 50 μl) were injected sequentially after washing of the chip with binding buffer. The ΔRU for activator binding to TBP and that for holoenzyme binding to activator bound to TBP are shown.Mol Cell Biol. ):.Publication TypesMeSH TermsSubstancesFull Text SourcesOther Literature SourcesMolecular Biology DatabasesMiscellaneous
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):2967-76.An activator binding module of yeast RNA polymerase II holoenzyme.1, , , , .1Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University College of Medicine, Kangnam-ku, Seoul 135-230, Korea.AbstractThe Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system. This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter. Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays. In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme. These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity.PMID:
[PubMed - indexed for MEDLINE] PMCID: PMC84091 Subunit compositions of mutant holoenzymes. (A) Immunoprecipitation analysis. Holoenzymes (MonoQ fractions) were prepared from wild-type (YCL10; W), rgr1Δ2 (DY2010; R), gal11Δ (HS301; G), hrs1Δ (SSAB-2CF; H), and srb5Δ (CTY153; S) strains and immunoprecipitated with anti-Rgr1 antibody–beads as described in Materials and Methods. Proteins were resolved on an SDS–10% polyacrylamide gel and visualized by silver staining. The positions of core polymerase subunits (Rpb) and Mediator components are indicated at the left and right, respectively. (B) Immunoblot analysis. Wild-type (W) and mutant (R, rgr1Δ2; S, srb5Δ; G, gal11Δ; H, hrs1Δ) holoenzymes (MonoQ fractions) were subjected to immunoblot analysis with antisera specific for the PolII and Mediator components indicated between the panels. Mol Cell Biol. ):.In vitro transcription of mutant holoenzymes. Wild-type and mutant holoenzymes (700 ng each) containing the same levels of nonspecific transcriptional elongation activities were analyzed for their promoter-specific transcriptional activities in the presence of the indicated activators in an in vitro transcription system reconstituted with pure general transcription factors and other supplements as described previously (21). RNA transcripts from templates containing either the Gal4 binding site (GAL4:G-) or the GCN4 binding site (GCN4:G-) are indicated. (A) Transcriptional defects of rgr1Δ2 and srb5Δ holoenzymes. The specifically initiated transcripts from reactions that contained wild-type (W), rgr1Δ2 (R), and srb5Δ (S) holoenzymes in the absence (none) or presence of activator protein (Gal4VP16 or GCN4; 30 ng each) are shown. (B) Transcriptional defects of gal11Δ and hrs1Δ holoenzymes. The specifically initiated transcripts from reactions that contained wild-type, gal11Δ, and hrs1Δ holoenzymes in the absence (-) or presence of activator protein (Gal4VP16 [V] or GCN4 [N]; 30 ng each) are shown.Mol Cell Biol. ):.TFIIH-dependent CTD phosphorylation of mutant holoenzymes. (A) Core polymerase (core-polII, 0.3 μg) and holoenzyme (Holo-polII, 0.9 μg) were incubated with [γ-32P]ATP and without (-) or with (+) the indicated supplements (Gal4VP16 [Gal4VP, 30 ng], TBP [50 ng], TFIIB [50 ng], TFIIE [60 ng], TFIIH [60 ng], TFIIF [F, 20 ng], and DNA [pJJ470, 200 ng]) in transcription buffer for 10 min (holoenzyme) or 40 min (core polymerase) at 25°C. Phosphorylated Rpb1 was analyzed by SDS-PAGE (7.5% gel) and visualized by autoradiography. (B) CTD phosphorylation of mutant holoenzymes during Gal4VP16-mediated transcriptional activation. The degrees of CTD phosphorylation of the indicated holoenzymes (900 ng) under various transcription reaction conditions are shown. CTD phosphorylation by TFIIH (60 ng) only in the presence of the DNA template (pJJ470, 200 lane H) and under basal (lane B) or activated (Gal4VP16 mediated, lane A) transcription conditions is shown. (C) CTD phosphorylation of mutant holoenzymes during GCN4-mediated transcriptional activation. CTD of wild-type (W), rgr1Δ2 (R), srb5Δ (S), gal11Δ (G), and hrs1Δ (H) holoenzymes was phosphorylated by TFIIH under basal or activated (GCN4-mediated) transcription conditions.Mol Cell Biol. ):.Interactions of transcriptional activators with wild-type and mutant holoenzymes. (A, B, and D) Plotted is the change in the refractory index units (RU) versus time after injection of the indicated holoenzymes onto an activator-immobilized Biosensor chip. (A) SPR analysis of the interactions between Gal4VP16 and wild-type and rgr1Δ2 holoenzymes (70 μl of a MonoQ fraction at a concentration of 200 μg/ml). (B) SPR analysis of the interactions between wild-type (W) and hrs1Δ (H) holoenzymes (50 μl of a MonoQ fraction at a concentration of 250 μg/ml) and GST-VP16 or GCN4. (C) Coimmunoprecipitation (I.P.) of Gal4VP16 with wild-type (W) and mutant (R, rgr1Δ2) holoenzymes followed by Western blotting. Each holoenzyme (MonoQ fraction, 10 μg) was mixed with Gal4VP16 (300 ng) and immunoprecipitated with anti-Rgr1 antibody–beads (see Materials and Methods). In order to measure the amounts of precipitated Gal4VP16 and holoenzymes, we included equimolar amounts of recombinant Gal4VP16 and Med6 proteins (r-P; Med6, 80 Gal4VP16, 60 ng) as quantitative standards. Immunoblot analyses with antisera specific for the proteins indicated to the right of the panel are shown. (D) SPR analysis of the interactions between wild-type (W, 150 μg/ml) and srb5Δ (S, 450 μg/ml) holoenzymes and an activator (GST-VP16). Due to the low specific concentration of the srb5Δ holoenzyme in the MonoQ fraction (one-fifth the wild-type level), a threefold-larger amount of srb5Δ MonoQ fraction was injected to supply an amount of the srb5Δ holoenzyme comparable to that of the wild-type enzyme.Mol Cell Biol. ):.Direct interaction of Gal11 with acidic activators. (A) Far-Western analysis of holoenzymes with Gal4VP16. Wild-type (W) and gal11Δ (G) holoenzymes (immunopurified from a MonoQ fraction with an anti-Rgr1 antibody column, ~2 μg each) were resolved on an SDS–10% polyacrylamide gel and transferred to a nitrocellulose membrane. The proteins were renatured and allowed to bind to 32P-labeled Gal4VP16 as described in Materials and Methods. The position of the Gal11 protein was revealed by Western analysis of the same blot with the use of anti-Gal11 antiserum (αGal11). (B) Column binding assay for interactions between Gal11 and acidic activators. Agarose beads conjugated with purified GST or GST-Gal11 (5 μg each) were incubated overnight at 4°C with 10 μl of in vitro-translated, 35S-labeled Gal4VP16, Gal4VP16Δ456FP442, or GCN4. After extensive washing, the beads were boiled in SDS sample buffer, and the proteins were resolved by SDS–12.5% PAGE and subjected to autoradiography. Load represents 10% of the amount of in vitro-translated products loaded on the beads.Mol Cell Biol. ):.Simultaneous interactions of an activator with a holoenzyme and TBP. The ΔRU during the time course of the SPR analysis with a TBP-immobilized Biosensor chip is shown. Gal4VP16 (30 μg/ml, 30 μl) and wild-type holoenzyme (Holo-polII; 300 μg/ml, 50 μl) were injected sequentially after washing of the chip with binding buffer. The ΔRU for activator binding to TBP and that for holoenzyme binding to activator bound to TBP are shown.Mol Cell Biol. ):.Publication TypesMeSH TermsSubstancesFull Text SourcesOther Literature SourcesMolecular Biology DatabasesMiscellaneous
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External link. Please review our .Complexation of the tissue plasminogen activator protease with benz...
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1996 Mar 12;35(10):3270-6.Complexation of the tissue plasminogen activator protease with benzamidine-type inhibitors: interference by the kringle 2 module.1, , , , .1Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.AbstractWell-resolved high-field 1H NMR signals between -0.1 and -0.7 ppm afford convenient probes to monitor the conformational state of the tissue plasminogen activator (tPA) protease, modulated by covalent inhibitor binding or activation cleavage [Hu, C.-K., Kohnert, U., Wilhelm, O., Fischer, S., & Llinas, M. (1994) Biochemistry 33, ]. We have investigated recombinant BM 06.022 (a domain-deletion variant mutant from Escherichia coli comprising the kringle 2 and protease modules) and protease constructs of tPA in both single-chain (sc) and two-chain (tc) forms. The two proteins were studied when confronted with the noncovalent (i.e., reversible) active site inhibitors benzamidine and a series of bisbenzamidine derivatives: 2,5-bis(4-amidinobenzylidene)cyclopentanone, 2,6-bis(4-amidinobenzylidene)cyclohexanone, 2,7-bis(4-amidinobenzylidene)cycloheptanone, and 2,8-bis(4-amidino- benzylidene)cyclooctanone. At pH* 4.6, the 1H NMR spectrum is sensitive to complexation of the protease module with the various effectors. The amplitude of the inhibitor-shifted resonances is more pronounced for the tc-protease than for the sc-protease, suggesting that access of inhibitors to the protease catalytic site is facilitated upon conversion to the tc form. The effects detected by the NMR spectrum suggest a biphasic process, involving stronger (primary) and weaker (secondary) bindings to a single protease active site. Binding to the protease module in tc-BM 06.022 essentially generates the same spectral characteristics as detected upon binding to the isolated tc-protease construct. In contrast, a negligible perturbation by the inhibitors is observed on the (sc) BM 06.022. Hence, in the intact BM 06.022 the kringle 2 is structurally coupled to the protease module thus interfering with inhibitor molecules from accessing the protease active site. These domain-domain interactions relax upon conversion to the catalytically active tc form, thus decoupling the kringle 2 from the protease module in BM 06.022 while simultaneously exposing the active site to become accessible to effectors or substrates.PMID: 8605163
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