1. Function of a Eukaryotic Transcription Activator during the Transcription Cycle 
James Fishburn, Neeman Mohibullah, Steven Hahn 
Molecular Cell 
Volume 18, Issue 3, Pages (April 2005)
DOI: /j.molcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1
Site-Specific Photocrosslinking of the Gcn4 Central Activating Region during Transcription Activation
(A) The sequence of the Gcn4 central activating region with cysteine substitutions indicated.
(B) mGcn4 with 125I-PEAS attached to the indicated positions were tested for activity in multiround transcription at the HIS4 promoter. mGcn4 with no added Cys residues (wt) is also shown.
(C) Crosslinking in PICs analyzed by SDS-PAGE and phosphorimager. PICs were formed for 40 min on an immobilized HIS4 promoter template (Ranish et al., 1999) containing a single Gcn4 binding site in the presence of the indicated mGcn4-125I-PEAS derivatives and, where indicated, UV crosslinked for 1 min and reduced with DTT. Three crosslinking targets and protein size standards are indicated.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Identification of the mGcn4 Crosslinking Targets
(A) Identification of crosslinking targets by SDS-PAGE mobility shift. mGcn4 C132-PEAS and nuclear extracts from strains containing triple Flag-tagged Taf12, Gal11, or a nontagged strain were used for PIC formation and UV crosslinked as in Figure 1. Tagged and nontagged crosslinked targets are indicated on right.
(B and C) Identification of crosslinking targets by immune precipitation. PICs were formed and crosslinked as in (A) using the indicated Flag-tagged extracts. Crosslinked PICs were reduced with DTT and disrupted with 1 M potassium acetate, and soluble proteins were precipitated with anti-Flag Sepharose and analyzed by SDS-PAGE. In (C), the observed ratio of labeled Taf12/Tra1 is indicated.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Specificity of Crosslinking and Interactions of Activator during the Transcription Cycle
(A) Crosslinking at promoters with multiple Gcn4 binding sites. Crosslinking was performed as in Figure 1 at HIS4 promoters containing 1, 2, or 3 Gcn4 upstream binding sites in the orientations indicated by arrows. Crosslinked factors and molecular size standards are indicated.
(B) Crosslinking of activator during PIC formation. An immobilized HIS4 template with prebound mGcn4 was incubated with nuclear extract for the indicated times. PICs were treated with UV and DTT as indicated. Several background bands are observed in both the UV treated and untreated reactions.
(C) Comparison of mGcn4 crosslinking in the PIC, Scaffold, and second round PICs. Crosslinking in PICs was performed as in Figure 1 using untagged nuclear extract. Scaffold complexes were formed by addition of ATP, followed by UV crosslinking (Yudkovsky et al., 2000). Second round PICs were formed by addition of nuclear extract from an srb2Δ strain to scaffold complexes (Yudkovsky et al., 2000) followed by UV crosslinking for 1 min. Western analysis was used to confirm the formation of Scaffold or 2° PICs (not shown).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4
The Role of Gal11 in mGcn4 Crosslinking and Transcription Activation In Vitro
(A) Gcn4 crosslinking in the absence of Gal11. PICs were formed on immobilized templates using Gcn4 C132-PEAS and either wild-type or gal11Δ extract, crosslinked, and analyzed as in Figure 1. Crosslinking targets and protein size markers are indicated.
(B) In vitro transcription in the absence of Gal11. Comparison of wild-type and gal11Δ nuclear extract for activity in single and multiround transcription. Transcription was performed on HIS4 promoter plasmid templates containing a single Gcn4 binding site for either 3 min or 40 min with mGcn4 added as indicated. All experiments were repeated three times and the average fold activation and standard deviation (SD) is indicated.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 The Gcn4-Tra1 Interaction Is Essential for Activation
(A) Sequence of the Gcn4 central activating region with the C132 and triple alanine substitution mutation indicated.
(B) The mGcn4 mutant is defective for transcription activation in vitro. mGcn4, mGcn4 C132, and mGcn4 C132 triple ala mutant derivatives were assayed for in vitro activity using an immobilized HIS4 promoter. Transcription was repeated three times and the fold activation and standard deviation is indicated.
(C) The mGcn4 triple ala mutant is defective for interaction with Tra1. PICs were formed using either mGcn4 C132-PEAS or the mGcn4 C132-PEAS triple ala mutant and crosslinked as in Figure 1. Crosslinking products are indicated. The results of two independent experiments are shown, fractionated on different SDS NuPAGE systems (Invitrogen) to better resolve the different size crosslinking products.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
SAGA, but Not NuA4 or TFIID, Is Required for Activation In Vitro
Yeast nuclear extracts were immune depleted for either SAGA, TFIID, or NuA4 using extracts containing triple Flag tags on one or two subunits specific for each complex. Extracts were assayed for in vitro transcription on HIS4 plasmid templates with mGcn4. SAGA-depleted extracts (Spt7, Ada1 double-tagged strain) assayed for (A) single or (B) multiround transcription. Flag-tagged (F) or TAP-tagged (T) purified SAGA was added as indicated. (C) NuA4-depleted extract (Esa1-tagged strain) assayed for multiround transcription. TFIID-depleted extract (Taf3, Taf7 double-tagged strain) assayed for (D) single or (E) multiround transcription. For all in vitro transcription in this figure, average fold activation from two experiments is indicated along with standard deviation (SD).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions