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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

Tracking of the TFIID inhibitory activity to a tin-containing complex.(A) Scheme of the recrystallization procedure provided by the vender for the resynthesis of Princeton OSSK_462080. (B) Detection of robust TFIID inhibitory activity within the recrystallization leftover. For comparison, the corresponding ‘pure’ chemical (Princeton OSSK_462080 large-scale resynthesis) only inhibited TFIID-directed transcription by twofold at 10 µM (4.6 µg/ml; 10-fold at 100 µM) (Figure 3—figure supplement 1B). (C) Recreation of the TFIID inhibitory activity by mimicking the recrystallization procedure. Top, the scheme. Inactive compound 2 (LifeChem F1566-0338 large-scale resynthesis), tin(II) chloride, and acetate acid were dissolved in isopropanol in a sealed container with atmosphere air and incubated at 83°C. Samples were taken at different time point and assayed for activity (with a final concentration equivalent to 10 µM elemental tin), as shown on the bottom. (D) Comparing different tin compounds in recreating the inhibitory activity, in the presence (left) or absence (right) of the inactive mother molecule (compound 2). The reaction conditions are as described in (C). Tin (II or IV) oxides were used as suspension in corresponding reactions.DOI:http://dx.doi.org/10.7554/eLife.07777.008
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fig3s2: Tracking of the TFIID inhibitory activity to a tin-containing complex.(A) Scheme of the recrystallization procedure provided by the vender for the resynthesis of Princeton OSSK_462080. (B) Detection of robust TFIID inhibitory activity within the recrystallization leftover. For comparison, the corresponding ‘pure’ chemical (Princeton OSSK_462080 large-scale resynthesis) only inhibited TFIID-directed transcription by twofold at 10 µM (4.6 µg/ml; 10-fold at 100 µM) (Figure 3—figure supplement 1B). (C) Recreation of the TFIID inhibitory activity by mimicking the recrystallization procedure. Top, the scheme. Inactive compound 2 (LifeChem F1566-0338 large-scale resynthesis), tin(II) chloride, and acetate acid were dissolved in isopropanol in a sealed container with atmosphere air and incubated at 83°C. Samples were taken at different time point and assayed for activity (with a final concentration equivalent to 10 µM elemental tin), as shown on the bottom. (D) Comparing different tin compounds in recreating the inhibitory activity, in the presence (left) or absence (right) of the inactive mother molecule (compound 2). The reaction conditions are as described in (C). Tin (II or IV) oxides were used as suspension in corresponding reactions.DOI:http://dx.doi.org/10.7554/eLife.07777.008

Mentions: In an effort to perform a structure-activity relationship analysis, we resynthesized compound 1 in-house and were surprised to find that the resynthesized compound was completely inactive in the transcription assay (Figure 3A). By comparing three batches of an analog compound (2) with varying levels of inhibitory activity (Figure 3—figure supplement 1), we found that the inhibitory activity correlated with levels of a tin-containing material detected by elemental analysis (Figure 3B). This material is likely derived from tin(II) chloride (SnCl2) added as an anti-oxidant in the final recrystallization step in a subset of the commercially supplied samples (Figure 3—figure supplement 2). After excluding most common tin-containing compounds as candidates, we found that tin(IV) oxochloride, prepared by any of several established routes (Dehnicke, 1961; Messin and Janierdubry, 1979; Sakurada et al., 2000), consistently reproduced the specific inhibition of TFIID-directed transcription (Figure 3C and Figure 3—figure supplement 3). Dose-response titration revealed a Hill coefficient of ∼1, suggesting a non-cooperative binding of this chemical to its biological target (Figure 3D). This compound, which consists of tin, bridging oxygen, and chlorine ligands, may form ladder-like clustered structures and coordinate to atoms with lone electron pairs, such as the nitrogen in pyridine (Dehnicke, 1961; Messin and Janierdubry, 1979; Holmes et al., 1987; Sakurada et al., 2000), or as is perhaps more functionally relevant, the imidazole groups of histidine residues in proteins (Figure 3—figure supplement 3E). We concluded that the tin(IV) oxochloride-derived cluster is the ingredient within the active commercial supplies responsible for the TFIID-specific transcriptional inhibitory activity.10.7554/eLife.07777.006Figure 3.Identification of a tin(IV) oxochloride-derived cluster as the TFIID-specific transcription inhibitor.


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)

Tracking of the TFIID inhibitory activity to a tin-containing complex.(A) Scheme of the recrystallization procedure provided by the vender for the resynthesis of Princeton OSSK_462080. (B) Detection of robust TFIID inhibitory activity within the recrystallization leftover. For comparison, the corresponding ‘pure’ chemical (Princeton OSSK_462080 large-scale resynthesis) only inhibited TFIID-directed transcription by twofold at 10 µM (4.6 µg/ml; 10-fold at 100 µM) (Figure 3—figure supplement 1B). (C) Recreation of the TFIID inhibitory activity by mimicking the recrystallization procedure. Top, the scheme. Inactive compound 2 (LifeChem F1566-0338 large-scale resynthesis), tin(II) chloride, and acetate acid were dissolved in isopropanol in a sealed container with atmosphere air and incubated at 83°C. Samples were taken at different time point and assayed for activity (with a final concentration equivalent to 10 µM elemental tin), as shown on the bottom. (D) Comparing different tin compounds in recreating the inhibitory activity, in the presence (left) or absence (right) of the inactive mother molecule (compound 2). The reaction conditions are as described in (C). Tin (II or IV) oxides were used as suspension in corresponding reactions.DOI:http://dx.doi.org/10.7554/eLife.07777.008
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Related In: Results  -  Collection

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fig3s2: Tracking of the TFIID inhibitory activity to a tin-containing complex.(A) Scheme of the recrystallization procedure provided by the vender for the resynthesis of Princeton OSSK_462080. (B) Detection of robust TFIID inhibitory activity within the recrystallization leftover. For comparison, the corresponding ‘pure’ chemical (Princeton OSSK_462080 large-scale resynthesis) only inhibited TFIID-directed transcription by twofold at 10 µM (4.6 µg/ml; 10-fold at 100 µM) (Figure 3—figure supplement 1B). (C) Recreation of the TFIID inhibitory activity by mimicking the recrystallization procedure. Top, the scheme. Inactive compound 2 (LifeChem F1566-0338 large-scale resynthesis), tin(II) chloride, and acetate acid were dissolved in isopropanol in a sealed container with atmosphere air and incubated at 83°C. Samples were taken at different time point and assayed for activity (with a final concentration equivalent to 10 µM elemental tin), as shown on the bottom. (D) Comparing different tin compounds in recreating the inhibitory activity, in the presence (left) or absence (right) of the inactive mother molecule (compound 2). The reaction conditions are as described in (C). Tin (II or IV) oxides were used as suspension in corresponding reactions.DOI:http://dx.doi.org/10.7554/eLife.07777.008
Mentions: In an effort to perform a structure-activity relationship analysis, we resynthesized compound 1 in-house and were surprised to find that the resynthesized compound was completely inactive in the transcription assay (Figure 3A). By comparing three batches of an analog compound (2) with varying levels of inhibitory activity (Figure 3—figure supplement 1), we found that the inhibitory activity correlated with levels of a tin-containing material detected by elemental analysis (Figure 3B). This material is likely derived from tin(II) chloride (SnCl2) added as an anti-oxidant in the final recrystallization step in a subset of the commercially supplied samples (Figure 3—figure supplement 2). After excluding most common tin-containing compounds as candidates, we found that tin(IV) oxochloride, prepared by any of several established routes (Dehnicke, 1961; Messin and Janierdubry, 1979; Sakurada et al., 2000), consistently reproduced the specific inhibition of TFIID-directed transcription (Figure 3C and Figure 3—figure supplement 3). Dose-response titration revealed a Hill coefficient of ∼1, suggesting a non-cooperative binding of this chemical to its biological target (Figure 3D). This compound, which consists of tin, bridging oxygen, and chlorine ligands, may form ladder-like clustered structures and coordinate to atoms with lone electron pairs, such as the nitrogen in pyridine (Dehnicke, 1961; Messin and Janierdubry, 1979; Holmes et al., 1987; Sakurada et al., 2000), or as is perhaps more functionally relevant, the imidazole groups of histidine residues in proteins (Figure 3—figure supplement 3E). We concluded that the tin(IV) oxochloride-derived cluster is the ingredient within the active commercial supplies responsible for the TFIID-specific transcriptional inhibitory activity.10.7554/eLife.07777.006Figure 3.Identification of a tin(IV) oxochloride-derived cluster as the TFIID-specific transcription inhibitor.

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