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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

Small-molecule microarray screening for TFIID-specific modulators.(A) Screening schematic: chemicals of diverse structure were covalently attached to a functionalized glass surface, and incubated with protein of interest, the binding of which was indicated by primary antibodies recognized by a specific fluorescently (Cy5, red) labeled secondary body. (B) Representative images of an area of arrays probed with bovine serum albumin (BSA, control), Drosophila (d) TFIID, human (h) TFIID, TFIIH (control), and Pol II (control) in combination with specified primary antibodies. Yellow arrows denote the lead compound (1, ChemDiv 7241-4207). The images were scanned at 532 nm (green, for reference spots) and 635 nm (red, for antibody signal). (C) Background subtracted average signal in arbitrary unit (A.U.) in the Cy5 fluorescent channel picked up by Drosophila (left) or human (right) TFIID is plotted against their respective BSA controls. Yellow circles depict the data points of the lead compound.DOI:http://dx.doi.org/10.7554/eLife.07777.003
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fig1: Small-molecule microarray screening for TFIID-specific modulators.(A) Screening schematic: chemicals of diverse structure were covalently attached to a functionalized glass surface, and incubated with protein of interest, the binding of which was indicated by primary antibodies recognized by a specific fluorescently (Cy5, red) labeled secondary body. (B) Representative images of an area of arrays probed with bovine serum albumin (BSA, control), Drosophila (d) TFIID, human (h) TFIID, TFIIH (control), and Pol II (control) in combination with specified primary antibodies. Yellow arrows denote the lead compound (1, ChemDiv 7241-4207). The images were scanned at 532 nm (green, for reference spots) and 635 nm (red, for antibody signal). (C) Background subtracted average signal in arbitrary unit (A.U.) in the Cy5 fluorescent channel picked up by Drosophila (left) or human (right) TFIID is plotted against their respective BSA controls. Yellow circles depict the data points of the lead compound.DOI:http://dx.doi.org/10.7554/eLife.07777.003

Mentions: We screened a library of ∼10,000 organic compounds for binders to metazoan TFIID using a small-molecule microarray platform (Casalena et al., 2012). In this screen, chemicals of diverse structures were printed on a functionalized glass surface and the binding of TFIID was detected by specific antibodies (Figure 1A). We identified one compound (1, ChemDiv 7241-4207) that reproducibly and selectively bound to both Drosophila and human TFIID (Figure 1B,C). As controls, no binding to the antibodies or two other multi-subunit complexes of the human Pol II core transcription machinery, TFIIH and Pol II, was observed in counter screenings (Figure 1B).10.7554/eLife.07777.003Figure 1.Small-molecule microarray screening for TFIID-specific modulators.


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)

Small-molecule microarray screening for TFIID-specific modulators.(A) Screening schematic: chemicals of diverse structure were covalently attached to a functionalized glass surface, and incubated with protein of interest, the binding of which was indicated by primary antibodies recognized by a specific fluorescently (Cy5, red) labeled secondary body. (B) Representative images of an area of arrays probed with bovine serum albumin (BSA, control), Drosophila (d) TFIID, human (h) TFIID, TFIIH (control), and Pol II (control) in combination with specified primary antibodies. Yellow arrows denote the lead compound (1, ChemDiv 7241-4207). The images were scanned at 532 nm (green, for reference spots) and 635 nm (red, for antibody signal). (C) Background subtracted average signal in arbitrary unit (A.U.) in the Cy5 fluorescent channel picked up by Drosophila (left) or human (right) TFIID is plotted against their respective BSA controls. Yellow circles depict the data points of the lead compound.DOI:http://dx.doi.org/10.7554/eLife.07777.003
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4582147&req=5

fig1: Small-molecule microarray screening for TFIID-specific modulators.(A) Screening schematic: chemicals of diverse structure were covalently attached to a functionalized glass surface, and incubated with protein of interest, the binding of which was indicated by primary antibodies recognized by a specific fluorescently (Cy5, red) labeled secondary body. (B) Representative images of an area of arrays probed with bovine serum albumin (BSA, control), Drosophila (d) TFIID, human (h) TFIID, TFIIH (control), and Pol II (control) in combination with specified primary antibodies. Yellow arrows denote the lead compound (1, ChemDiv 7241-4207). The images were scanned at 532 nm (green, for reference spots) and 635 nm (red, for antibody signal). (C) Background subtracted average signal in arbitrary unit (A.U.) in the Cy5 fluorescent channel picked up by Drosophila (left) or human (right) TFIID is plotted against their respective BSA controls. Yellow circles depict the data points of the lead compound.DOI:http://dx.doi.org/10.7554/eLife.07777.003
Mentions: We screened a library of ∼10,000 organic compounds for binders to metazoan TFIID using a small-molecule microarray platform (Casalena et al., 2012). In this screen, chemicals of diverse structures were printed on a functionalized glass surface and the binding of TFIID was detected by specific antibodies (Figure 1A). We identified one compound (1, ChemDiv 7241-4207) that reproducibly and selectively bound to both Drosophila and human TFIID (Figure 1B,C). As controls, no binding to the antibodies or two other multi-subunit complexes of the human Pol II core transcription machinery, TFIIH and Pol II, was observed in counter screenings (Figure 1B).10.7554/eLife.07777.003Figure 1.Small-molecule microarray screening for TFIID-specific modulators.

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