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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

Discrepancy between organic compound structures and the transcription inhibitor activity of commercial compounds.Transcription inhibition by lead compound 1 (A) and its analog 2 (B) from different sources. Because the actual inhibitory activity was found to be from minor contaminations unrelated to the assigned chemical structure, the reagents are named by an abbreviation of the vender name followed by the catalog number (or as ‘in-house’ preparation). ChemDiv 7241-4207 (original purchase, lead compound 1) was also used in (B) as a positive control for transcription inhibition. By analytical organic chemistry standards (nuclear magnetic resonance [NMR] and mass spectrometry), all these compounds are >95% ‘pure’. The three supplies of analog compound 2 subjected to elemental analysis (Figure 3B) are highlighted with colored stars (*) in (B), and their high-resolution NMR analysis results are shown in panel C (left). (C) (Right) is a hTFIID-directed transcription assay showing that the minor dimethylformamide (DMF) solvent contamination detected in the active materials by NMR as two chemical shifts between 2.5 and 3.0 ppm is irrelevant to the inhibitory activity.DOI:http://dx.doi.org/10.7554/eLife.07777.007
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fig3s1: Discrepancy between organic compound structures and the transcription inhibitor activity of commercial compounds.Transcription inhibition by lead compound 1 (A) and its analog 2 (B) from different sources. Because the actual inhibitory activity was found to be from minor contaminations unrelated to the assigned chemical structure, the reagents are named by an abbreviation of the vender name followed by the catalog number (or as ‘in-house’ preparation). ChemDiv 7241-4207 (original purchase, lead compound 1) was also used in (B) as a positive control for transcription inhibition. By analytical organic chemistry standards (nuclear magnetic resonance [NMR] and mass spectrometry), all these compounds are >95% ‘pure’. The three supplies of analog compound 2 subjected to elemental analysis (Figure 3B) are highlighted with colored stars (*) in (B), and their high-resolution NMR analysis results are shown in panel C (left). (C) (Right) is a hTFIID-directed transcription assay showing that the minor dimethylformamide (DMF) solvent contamination detected in the active materials by NMR as two chemical shifts between 2.5 and 3.0 ppm is irrelevant to the inhibitory activity.DOI:http://dx.doi.org/10.7554/eLife.07777.007

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)

Discrepancy between organic compound structures and the transcription inhibitor activity of commercial compounds.Transcription inhibition by lead compound 1 (A) and its analog 2 (B) from different sources. Because the actual inhibitory activity was found to be from minor contaminations unrelated to the assigned chemical structure, the reagents are named by an abbreviation of the vender name followed by the catalog number (or as ‘in-house’ preparation). ChemDiv 7241-4207 (original purchase, lead compound 1) was also used in (B) as a positive control for transcription inhibition. By analytical organic chemistry standards (nuclear magnetic resonance [NMR] and mass spectrometry), all these compounds are >95% ‘pure’. The three supplies of analog compound 2 subjected to elemental analysis (Figure 3B) are highlighted with colored stars (*) in (B), and their high-resolution NMR analysis results are shown in panel C (left). (C) (Right) is a hTFIID-directed transcription assay showing that the minor dimethylformamide (DMF) solvent contamination detected in the active materials by NMR as two chemical shifts between 2.5 and 3.0 ppm is irrelevant to the inhibitory activity.DOI:http://dx.doi.org/10.7554/eLife.07777.007
© Copyright Policy
Related In: Results  -  Collection

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

fig3s1: Discrepancy between organic compound structures and the transcription inhibitor activity of commercial compounds.Transcription inhibition by lead compound 1 (A) and its analog 2 (B) from different sources. Because the actual inhibitory activity was found to be from minor contaminations unrelated to the assigned chemical structure, the reagents are named by an abbreviation of the vender name followed by the catalog number (or as ‘in-house’ preparation). ChemDiv 7241-4207 (original purchase, lead compound 1) was also used in (B) as a positive control for transcription inhibition. By analytical organic chemistry standards (nuclear magnetic resonance [NMR] and mass spectrometry), all these compounds are >95% ‘pure’. The three supplies of analog compound 2 subjected to elemental analysis (Figure 3B) are highlighted with colored stars (*) in (B), and their high-resolution NMR analysis results are shown in panel C (left). (C) (Right) is a hTFIID-directed transcription assay showing that the minor dimethylformamide (DMF) solvent contamination detected in the active materials by NMR as two chemical shifts between 2.5 and 3.0 ppm is irrelevant to the inhibitory activity.DOI:http://dx.doi.org/10.7554/eLife.07777.007
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