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Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation.

Zhang Z, Boskovic Z, Hussain MM, Hu W, Inouye C, Kim HJ, Abole AK, Doud MK, Lewis TA, Koehler AN, Schreiber SL, Tjian R - Elife (2015)

Bottom Line: They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study.Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation.This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

View Article: PubMed Central - PubMed

Affiliation: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

ABSTRACT
Intrinsically disordered proteins/regions (IDPs/IDRs) are proteins or peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. In this study, we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

No MeSH data available.


Related in: MedlinePlus

(A) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations.(B) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207.DOI:http://dx.doi.org/10.7554/eLife.07777.013
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fig5s1: (A) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations.(B) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207.DOI:http://dx.doi.org/10.7554/eLife.07777.013

Mentions: (A) GST pull-down of double-stranded (ds) DNA oligonucleotides by GST-dTAF2 fragments. Top, the GST fusions (G: GST alone). Middle, the four DNA fragments tested, which are parts of the SCP1 (Juven-Gershon et al., 2006) used in this study (sequence shown, with the TATA, Inr, and DPE elements, from left to right, underscored). Bottom, DNA staining raw images (left) and bar representation of the bound/unbound DNA signals (right). (B) Binding of nuclease-treated GST-dTAF2 (1125–1221) to the lead compound 1 (ChemDiv 7241-4207) printed in quadruplicates in the microarray (yellow rectangle), and its rescue by a double-stranded (ds)DNA oligonucleotide (1 µg/ml) or heparin (2 µg/ml). GST antibody was used for detection. Recombinant GST-dTAF5 was used as a negative control. (C) DNase I footprinting assay on TFIID-promoter binding, in the presence of the lead compound (1, ChemDiv 7241-4207), a structural analog (2, Princeton OSSK_462080), or an unrelated, non-specific (NS) inhibitor (Maybridge BTB08547, see Figure 5—figure supplement 1B). Shown is the digestion product of the end-labeled DNA template separated by gel electrophoresis. The DNA template contains the SCP1. Black boxes depict the positions of the TATA, Inr, and DPE elements, respectively. Blue bracket indicates the ‘footprint’ of TFIID. For simplicity, only two bands (denoted by arrowheads), which were protected (A) or intensified (B) upon TFIID binding, were selected for quantification (right).


Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation.

Zhang Z, Boskovic Z, Hussain MM, Hu W, Inouye C, Kim HJ, Abole AK, Doud MK, Lewis TA, Koehler AN, Schreiber SL, Tjian R - Elife (2015)

(A) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations.(B) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207.DOI:http://dx.doi.org/10.7554/eLife.07777.013
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4582147&req=5

fig5s1: (A) DNase I footprinting assay of TFIID-promoter binding and its enhancement by the specific inhibitor at different salt concentrations.(B) A non-specific inhibitor (Maybridge BTB08574): its structure (top) and inhibition of both TFIID- and TBP-directed transcription (bottom). The lead compound used here was ChemDiv 7241-4207.DOI:http://dx.doi.org/10.7554/eLife.07777.013
Mentions: (A) GST pull-down of double-stranded (ds) DNA oligonucleotides by GST-dTAF2 fragments. Top, the GST fusions (G: GST alone). Middle, the four DNA fragments tested, which are parts of the SCP1 (Juven-Gershon et al., 2006) used in this study (sequence shown, with the TATA, Inr, and DPE elements, from left to right, underscored). Bottom, DNA staining raw images (left) and bar representation of the bound/unbound DNA signals (right). (B) Binding of nuclease-treated GST-dTAF2 (1125–1221) to the lead compound 1 (ChemDiv 7241-4207) printed in quadruplicates in the microarray (yellow rectangle), and its rescue by a double-stranded (ds)DNA oligonucleotide (1 µg/ml) or heparin (2 µg/ml). GST antibody was used for detection. Recombinant GST-dTAF5 was used as a negative control. (C) DNase I footprinting assay on TFIID-promoter binding, in the presence of the lead compound (1, ChemDiv 7241-4207), a structural analog (2, Princeton OSSK_462080), or an unrelated, non-specific (NS) inhibitor (Maybridge BTB08547, see Figure 5—figure supplement 1B). Shown is the digestion product of the end-labeled DNA template separated by gel electrophoresis. The DNA template contains the SCP1. Black boxes depict the positions of the TATA, Inr, and DPE elements, respectively. Blue bracket indicates the ‘footprint’ of TFIID. For simplicity, only two bands (denoted by arrowheads), which were protected (A) or intensified (B) upon TFIID binding, were selected for quantification (right).

Bottom Line: They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study.Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation.This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

View Article: PubMed Central - PubMed

Affiliation: Transcription Imaging Consortium, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States.

ABSTRACT
Intrinsically disordered proteins/regions (IDPs/IDRs) are proteins or peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. In this study, we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

No MeSH data available.


Related in: MedlinePlus