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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

DNase I footprinting assays monitoring PIC assembly and structural isomerization.(A) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). (B) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl2·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in Figure 6 legend.DOI:http://dx.doi.org/10.7554/eLife.07777.016
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fig7: DNase I footprinting assays monitoring PIC assembly and structural isomerization.(A) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). (B) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl2·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in Figure 6 legend.DOI:http://dx.doi.org/10.7554/eLife.07777.016

Mentions: To better understand how Pol II engagement might be inhibited by the chemical, we performed DNase I footprinting assays to directly examine the potential conformational isomerization events during PIC assembly. In this assay, various PIC components were incubated with the template DNA under conditions that would lead to optimal transcription output if all the other components were included. We found that, as expected, TFIID alone caused a footprint covering the Inr, DPE, and extending downstream (to ∼+55) (Figure 7A, lane 1 and 2). The addition of TFIIB enhanced protection over the upstream region (from the TATA box to the Inr), and this protection became further enhanced by the addition of TFIIF (lanes 3 and 4), consistent with the synergy between TBP and TFIIB binding to promoter DNA (Tsai and Sigler, 2000), and the stabilization of TFIIB binding by TFIIF (Luse, 2012). Interestingly, we observed significant changes in the footprint pattern upon the addition of Pol II (lane 5). These changes include (i) the exposure of some hypersensitive sites protected by the initial TFIID binding (such as position ∼+16, and the sites flanking the DPE), suggesting the release or unmasking of some DNA from the bound TFIID; (ii) emergence of new hypersensitive sites (such as position ∼+14), suggesting structural changes in the DNA trajectory itself caused by Pol II binding; (iii) reduction of the hypersensitive site induced by TFIID binding at the edge of the Inr, consistent with the release of some DNA from TFIID and/or the association of Pol II; and (iv) a strong and extended protection covering the upstream of the TATA box (∼−37) to the Inr, consistent with the engagement of Pol II and other PIC components with this region of the promoter. These results are also consistent with a previous report using a different promoter (Yakovchuk et al., 2010) that suggested some kind of a conformational isomerization at the promoter associated with Pol II engagement.10.7554/eLife.07777.016Figure 7.DNase I footprinting assays monitoring PIC assembly and structural isomerization.


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)

DNase I footprinting assays monitoring PIC assembly and structural isomerization.(A) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). (B) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl2·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in Figure 6 legend.DOI:http://dx.doi.org/10.7554/eLife.07777.016
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4582147&req=5

fig7: DNase I footprinting assays monitoring PIC assembly and structural isomerization.(A) Early steps of PIC assembly. Specified GTFs were incubated with the end-labeled DNA template, followed by DNase I digestion. Blue bracket highlights the TFIID footprint. The numbers are relative to the transcription start site (+1). (B) Arresting of the conformational isomerization. Top, the scheme. The inhibitor used here was SnOCl2·pyridine. Lanes 1 and 9 are 10 bp DNA ladder. Lanes 2 and 10 are digestion of naked DNA. The lower case ‘d’ and ‘f’ in lanes #11–14 reflect the use of less TFIID and TFIIF (together with the omission of spermidine and carrier nucleic acid in the reaction—see ‘Material and methods’ for detail). Letter abbreviations are explained in Figure 6 legend.DOI:http://dx.doi.org/10.7554/eLife.07777.016
Mentions: To better understand how Pol II engagement might be inhibited by the chemical, we performed DNase I footprinting assays to directly examine the potential conformational isomerization events during PIC assembly. In this assay, various PIC components were incubated with the template DNA under conditions that would lead to optimal transcription output if all the other components were included. We found that, as expected, TFIID alone caused a footprint covering the Inr, DPE, and extending downstream (to ∼+55) (Figure 7A, lane 1 and 2). The addition of TFIIB enhanced protection over the upstream region (from the TATA box to the Inr), and this protection became further enhanced by the addition of TFIIF (lanes 3 and 4), consistent with the synergy between TBP and TFIIB binding to promoter DNA (Tsai and Sigler, 2000), and the stabilization of TFIIB binding by TFIIF (Luse, 2012). Interestingly, we observed significant changes in the footprint pattern upon the addition of Pol II (lane 5). These changes include (i) the exposure of some hypersensitive sites protected by the initial TFIID binding (such as position ∼+16, and the sites flanking the DPE), suggesting the release or unmasking of some DNA from the bound TFIID; (ii) emergence of new hypersensitive sites (such as position ∼+14), suggesting structural changes in the DNA trajectory itself caused by Pol II binding; (iii) reduction of the hypersensitive site induced by TFIID binding at the edge of the Inr, consistent with the release of some DNA from TFIID and/or the association of Pol II; and (iv) a strong and extended protection covering the upstream of the TATA box (∼−37) to the Inr, consistent with the engagement of Pol II and other PIC components with this region of the promoter. These results are also consistent with a previous report using a different promoter (Yakovchuk et al., 2010) that suggested some kind of a conformational isomerization at the promoter associated with Pol II engagement.10.7554/eLife.07777.016Figure 7.DNase I footprinting assays monitoring PIC assembly and structural isomerization.

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