Limits...
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

TFIID dependency of the in vitro transcription assay and controls for the TFIID-specific inhibition.(A) Titration of hTFIID in the reconstituted transcription reaction. The reaction contains all other protein factors specified in Figure 2, except for the lead compound. (B) Titration of hTFIID and dTFIID and their response to the lead compound 1 ChemDiv 7241-4207. Asterisk (*) denotes quantifications affected by cross-over signal from a neighbor. (C) TFIID-specific transcription inhibition on mutant SCP1s (Juven-Gershon et al., 2006). These images were from a same gel with the same display setting as those shown in Figure 2B. Note that although the absolute signal from TFIID-directed transcription may be core promoter element-dependent, ∼1.5 µg/ml of lead compound 1 is always sufficient to cause ∼50% transcription inhibition. In contrast, TBP-directed transcription is rather constant.DOI:http://dx.doi.org/10.7554/eLife.07777.005
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4582147&req=5

fig2s1: TFIID dependency of the in vitro transcription assay and controls for the TFIID-specific inhibition.(A) Titration of hTFIID in the reconstituted transcription reaction. The reaction contains all other protein factors specified in Figure 2, except for the lead compound. (B) Titration of hTFIID and dTFIID and their response to the lead compound 1 ChemDiv 7241-4207. Asterisk (*) denotes quantifications affected by cross-over signal from a neighbor. (C) TFIID-specific transcription inhibition on mutant SCP1s (Juven-Gershon et al., 2006). These images were from a same gel with the same display setting as those shown in Figure 2B. Note that although the absolute signal from TFIID-directed transcription may be core promoter element-dependent, ∼1.5 µg/ml of lead compound 1 is always sufficient to cause ∼50% transcription inhibition. In contrast, TBP-directed transcription is rather constant.DOI:http://dx.doi.org/10.7554/eLife.07777.005

Mentions: To assess the effect of compound 1 on transcription, we developed an integrated functional assay consisting of a reconstituted human cell-free transcription system (Figure 2A). In this assay, a complete set of highly purified GTFs (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH; TFIIA is not required) plus Pol II was incubated with the lead compound first, followed by incubation with a promoter-containing DNA template for transcription. As a control, TBP was used in place of TFIID to support a ‘basal’ transcription that also requires the rest of the protein factors. We found that the commercially supplied compound 1 (ChemDiv 7241-4207) inhibited both Drosophila and human TFIID-directed transcription, but not transcription directed by TBP (Figure 2B and Figure 2—figure supplement 1A,B), suggesting a TAF-specific mechanism of inhibition. Further characterization indicated that this inhibition (i) is sensitive to the dose of TFIID used in the reaction (Figure 2—figure supplement 1B), (ii) can be alleviated by the addition of more TFIID after chemical treatment, but not by the addition of any other protein factors (Figure 2C), confirming that TFIID is the most likely target of inhibition in the reaction, and (iii) is insensitive to various mutations in core promoter elements (Figure 2—figure supplement 1C). Taken together, our transcription results suggest that the inhibitory activity specifically targets an evolutionarily conserved TAF subunit of TFIID that is required for a basic function of TFIID during Pol II transcription initiation in vitro.10.7554/eLife.07777.004Figure 2.TFIID-specific transcription inhibition in a reconstituted system.


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)

TFIID dependency of the in vitro transcription assay and controls for the TFIID-specific inhibition.(A) Titration of hTFIID in the reconstituted transcription reaction. The reaction contains all other protein factors specified in Figure 2, except for the lead compound. (B) Titration of hTFIID and dTFIID and their response to the lead compound 1 ChemDiv 7241-4207. Asterisk (*) denotes quantifications affected by cross-over signal from a neighbor. (C) TFIID-specific transcription inhibition on mutant SCP1s (Juven-Gershon et al., 2006). These images were from a same gel with the same display setting as those shown in Figure 2B. Note that although the absolute signal from TFIID-directed transcription may be core promoter element-dependent, ∼1.5 µg/ml of lead compound 1 is always sufficient to cause ∼50% transcription inhibition. In contrast, TBP-directed transcription is rather constant.DOI:http://dx.doi.org/10.7554/eLife.07777.005
© Copyright Policy
Related In: Results  -  Collection

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

fig2s1: TFIID dependency of the in vitro transcription assay and controls for the TFIID-specific inhibition.(A) Titration of hTFIID in the reconstituted transcription reaction. The reaction contains all other protein factors specified in Figure 2, except for the lead compound. (B) Titration of hTFIID and dTFIID and their response to the lead compound 1 ChemDiv 7241-4207. Asterisk (*) denotes quantifications affected by cross-over signal from a neighbor. (C) TFIID-specific transcription inhibition on mutant SCP1s (Juven-Gershon et al., 2006). These images were from a same gel with the same display setting as those shown in Figure 2B. Note that although the absolute signal from TFIID-directed transcription may be core promoter element-dependent, ∼1.5 µg/ml of lead compound 1 is always sufficient to cause ∼50% transcription inhibition. In contrast, TBP-directed transcription is rather constant.DOI:http://dx.doi.org/10.7554/eLife.07777.005
Mentions: To assess the effect of compound 1 on transcription, we developed an integrated functional assay consisting of a reconstituted human cell-free transcription system (Figure 2A). In this assay, a complete set of highly purified GTFs (TFIIB, TFIID, TFIIE, TFIIF, and TFIIH; TFIIA is not required) plus Pol II was incubated with the lead compound first, followed by incubation with a promoter-containing DNA template for transcription. As a control, TBP was used in place of TFIID to support a ‘basal’ transcription that also requires the rest of the protein factors. We found that the commercially supplied compound 1 (ChemDiv 7241-4207) inhibited both Drosophila and human TFIID-directed transcription, but not transcription directed by TBP (Figure 2B and Figure 2—figure supplement 1A,B), suggesting a TAF-specific mechanism of inhibition. Further characterization indicated that this inhibition (i) is sensitive to the dose of TFIID used in the reaction (Figure 2—figure supplement 1B), (ii) can be alleviated by the addition of more TFIID after chemical treatment, but not by the addition of any other protein factors (Figure 2C), confirming that TFIID is the most likely target of inhibition in the reaction, and (iii) is insensitive to various mutations in core promoter elements (Figure 2—figure supplement 1C). Taken together, our transcription results suggest that the inhibitory activity specifically targets an evolutionarily conserved TAF subunit of TFIID that is required for a basic function of TFIID during Pol II transcription initiation in vitro.10.7554/eLife.07777.004Figure 2.TFIID-specific transcription inhibition in a reconstituted system.

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