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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

Controls for reinitiation experiments.(A) Sarkosyl titration. Sarkosyl was added at the beginning of PIC assembly (a), immediately (within 30 s) before (b) or after (c) the addition of NTPs (top scheme), and the effect on transcription was detected by primer extension (bottom images). In blue are the normalized transcription signals. (B) Comparison of single-round transcription vs multiple-round transcription using Sarkosyl treatment. Left, the scheme. Right, the image of the primer extension products. The transcription signals were quantified, normalized, and fold of inhibition under each condition were calculated. 0.1% Sarkosyl was added within 30 s after NTPs to restrict transcription to a single round. (C) Sarkosyl control for the G-less cassette transcription. 0.02% Sarkosyl was added right before the addition of NTPs to prevent reinitiation. Black arrowheads points to the bands corresponding to the first, second, and third transcript synthesized from the same DNA templates, and their signals were quantified, normalized (the blue numbers), and plotted below the image.DOI:http://dx.doi.org/10.7554/eLife.07777.018
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fig8s1: Controls for reinitiation experiments.(A) Sarkosyl titration. Sarkosyl was added at the beginning of PIC assembly (a), immediately (within 30 s) before (b) or after (c) the addition of NTPs (top scheme), and the effect on transcription was detected by primer extension (bottom images). In blue are the normalized transcription signals. (B) Comparison of single-round transcription vs multiple-round transcription using Sarkosyl treatment. Left, the scheme. Right, the image of the primer extension products. The transcription signals were quantified, normalized, and fold of inhibition under each condition were calculated. 0.1% Sarkosyl was added within 30 s after NTPs to restrict transcription to a single round. (C) Sarkosyl control for the G-less cassette transcription. 0.02% Sarkosyl was added right before the addition of NTPs to prevent reinitiation. Black arrowheads points to the bands corresponding to the first, second, and third transcript synthesized from the same DNA templates, and their signals were quantified, normalized (the blue numbers), and plotted below the image.DOI:http://dx.doi.org/10.7554/eLife.07777.018

Mentions: We first performed transcription reactions with two DNA templates that produce transcripts of distinct lengths (Figure 8A). We incubated the first DNA template (DNA 1) with a complete set of protein factors to form a stable PIC, then added the second DNA template (DNA 2) to see whether the essential factors are still available. To constrain transcription to a single round so that we can compare de novo PIC assembly on the two templates, we added 0.1% Sarkosyl immediately after the addition of nucleoside triphosphates (NTPs) for RNA synthesis following previously reported procedures using crude nuclear extracts (Hawley and Roeder, 1985; Kadonaga, 1990), which is also validated in our highly purified system (Figure 8—figure supplement 1A). We found that the presence and pre-incubation of DNA 1 severely compromised (by 11–18-fold) transcription from DNA 2 (Figure 8A, compare lanes 2–4 with 7 for the short transcript, and lanes 9–11 with 14 for the long transcript), indicating that some limiting factors become stably committed to the first DNA template during initial PIC assembly. Adding more fresh TFIID (1 × equivalent to the initial dosage) immediately after DNA 2 restored transcription activity from DNA 2 by ∼fourfold (comparing lanes 4 with 5, and 11 with 12), suggesting that TFIID is likely one of the limiting and DNA template committed factors during de novo PIC assembly.10.7554/eLife.07777.017Figure 8.Template commitment of TFIID and its resistant to tin(IV) oxochloride inhibition during reinitiation.


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)

Controls for reinitiation experiments.(A) Sarkosyl titration. Sarkosyl was added at the beginning of PIC assembly (a), immediately (within 30 s) before (b) or after (c) the addition of NTPs (top scheme), and the effect on transcription was detected by primer extension (bottom images). In blue are the normalized transcription signals. (B) Comparison of single-round transcription vs multiple-round transcription using Sarkosyl treatment. Left, the scheme. Right, the image of the primer extension products. The transcription signals were quantified, normalized, and fold of inhibition under each condition were calculated. 0.1% Sarkosyl was added within 30 s after NTPs to restrict transcription to a single round. (C) Sarkosyl control for the G-less cassette transcription. 0.02% Sarkosyl was added right before the addition of NTPs to prevent reinitiation. Black arrowheads points to the bands corresponding to the first, second, and third transcript synthesized from the same DNA templates, and their signals were quantified, normalized (the blue numbers), and plotted below the image.DOI:http://dx.doi.org/10.7554/eLife.07777.018
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Related In: Results  -  Collection

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fig8s1: Controls for reinitiation experiments.(A) Sarkosyl titration. Sarkosyl was added at the beginning of PIC assembly (a), immediately (within 30 s) before (b) or after (c) the addition of NTPs (top scheme), and the effect on transcription was detected by primer extension (bottom images). In blue are the normalized transcription signals. (B) Comparison of single-round transcription vs multiple-round transcription using Sarkosyl treatment. Left, the scheme. Right, the image of the primer extension products. The transcription signals were quantified, normalized, and fold of inhibition under each condition were calculated. 0.1% Sarkosyl was added within 30 s after NTPs to restrict transcription to a single round. (C) Sarkosyl control for the G-less cassette transcription. 0.02% Sarkosyl was added right before the addition of NTPs to prevent reinitiation. Black arrowheads points to the bands corresponding to the first, second, and third transcript synthesized from the same DNA templates, and their signals were quantified, normalized (the blue numbers), and plotted below the image.DOI:http://dx.doi.org/10.7554/eLife.07777.018
Mentions: We first performed transcription reactions with two DNA templates that produce transcripts of distinct lengths (Figure 8A). We incubated the first DNA template (DNA 1) with a complete set of protein factors to form a stable PIC, then added the second DNA template (DNA 2) to see whether the essential factors are still available. To constrain transcription to a single round so that we can compare de novo PIC assembly on the two templates, we added 0.1% Sarkosyl immediately after the addition of nucleoside triphosphates (NTPs) for RNA synthesis following previously reported procedures using crude nuclear extracts (Hawley and Roeder, 1985; Kadonaga, 1990), which is also validated in our highly purified system (Figure 8—figure supplement 1A). We found that the presence and pre-incubation of DNA 1 severely compromised (by 11–18-fold) transcription from DNA 2 (Figure 8A, compare lanes 2–4 with 7 for the short transcript, and lanes 9–11 with 14 for the long transcript), indicating that some limiting factors become stably committed to the first DNA template during initial PIC assembly. Adding more fresh TFIID (1 × equivalent to the initial dosage) immediately after DNA 2 restored transcription activity from DNA 2 by ∼fourfold (comparing lanes 4 with 5, and 11 with 12), suggesting that TFIID is likely one of the limiting and DNA template committed factors during de novo PIC assembly.10.7554/eLife.07777.017Figure 8.Template commitment of TFIID and its resistant to tin(IV) oxochloride inhibition during reinitiation.

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