Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site VSports app下载. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2010 May;30(10):2376-90.
doi: 10.1128/MCB.01046-09. Epub 2010 Mar 22.

Mechanism of Mediator recruitment by tandem Gcn4 activation domains and three Gal11 activator-binding domains

Eric Herbig  1 , Linda Warfield, Lisa Fish, James Fishburn, Bruce A Knutson, Beth Moorefield, Derek Pacheco, Steven Hahn
Affiliations

Mechanism of Mediator recruitment by tandem Gcn4 activation domains and three Gal11 activator-binding domains

Eric Herbig et al. Mol Cell Biol. 2010 May.

Abstract

Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive VSports手机版. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions. .

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
The Gcn4 N-terminal activation domain cross-links to Taf12, Gal11, Tra1, and Sin4 in PICs. (A) In vitro transcription from the HIS4 promoter comparing basal transcription (lane 1) to transcription activated by the Gcn4 N-terminal activation domain (Gcn4 Δ119-208) or with this Gcn4 derivative linked to the PEAS cross-linker at the indicated positions (lanes 3 to 8). (B) Photo-cross-linking in PICs using Gcn4-PEAS derivatives from panel A showing cross-linking to five proteins. Tra1* is a presumed breakdown product of Tra1. (C) Cross-linking in PICs assembled on either naked DNA or chromatin (Chrom)-assembled templates as indicated. Acetyl-CoA (AcCoA) was added as shown. (D, left side) Depletion or mock depletion of NuA4 from nuclear extracts containing HA-tagged Esa1 assayed by Western blotting. (D, right side) Transcription assays on chromatin or nonchromatin templates using Esa1-depleted or mock-depleted extracts. Acetyl-CoA was added to all reaction mixtures. The values to the left of panels B and C are molecular sizes in kilodaltons.
FIG. 2.
FIG. 2.
Identification of Gcn4 N-terminal activation domain cross-linking products. (A) Cross-linking reaction in PICs using nuclear extracts from the wild type (WT) (lane 1), Taf12-Flag (lane 2), Gal11-Flag (lane 3), or a Gal11 deletion strain visualized by PhosphorImager analysis probing for 125I on the PEAS cross-linker. The mobility shift indicates the identity of the cross-linked factor. (B) Immunoprecipitation (IP) of SAGA and NuA4 after cross-linking in PICs using a Flag tag on Ada1 (lane 1) or Esa1 (lane 2). (C) Cross-linking and immunoprecipitation of Mediator subunits using a Flag tag on Sin4 (lane 3) or Gal11 (lane 4) visualized by PhosphorImager. (D) Anti-Flag Western blotting of the gel shown in panel C indicating the identity of labeled cross-linked polypeptides. The values to the left of panels A to C are molecular sizes in kilodaltons.
FIG. 3.
FIG. 3.
Activity of the individual and combined Gcn4 activation domains and coactivator dependence of Gcn4-regulated genes. (A) Strain SHY823 containing wild-type (WT) Gcn4 or Gcn4 with a single functional N-terminal (NTAD) or central (CAD) activation domain (pSH940, pSH942, or pSH943) was treated with SM as indicated. mRNA levels from several Gcn4-dependent genes were measured by RT-qPCR. Error bars represent the standard error of the mean. (B) Strain SHY822 containing GCN4 (pSH828) and either GAL11 (pEH30) or the pRS316 vector lacking GAL11 was induced with SM as indicated, and mRNA was quantitated by RT-qPCR. (C) The indicated strains were induced with SM, and mRNA was quantitated by RT-qPCR. (D) Strains EHY41 and EHY42 containing either wild-type Taf12 or Taf12 with the Gcn4 activator-binding domain deleted were induced with SM as described above, and mRNA was quantitated by RT-qPCR.
FIG. 4.
FIG. 4.
Mapping of the Gcn4 activator-binding domains within Gal11. (A) Pulldown assay for initial localization of Gal11 activator-binding domains. Brackets above Gal11 indicate the three regions shown in this work to bind Gcn4 activation domains and the KIX domain. Bars below full-length Gcn4 indicate 35S-labeled Gal11 polypeptides produced by in vitro translation and tested for binding to both His6-Gcn4 and His6-Gcn4 residues 101 to 281 lacking the N-terminal activation domain and containing six Ala substitutions to inactivate the central activation domain. Gray bars indicate no Gcn4 binding compared to Gcn4 6× Ala, black bars indicate strong specific Gcn4 binding, and the hatched bar indicates weak specific binding. (B) Gcn4 derivatives containing either the Gcn4 N-terminal activation domain (AD) (residues 1 to 118) or the central activation domain (residues 101 to 134) were linked to FeBABE at the indicated cysteine residues and used to stimulate PIC formation on the immobilized HIS4 promoter using a Gal11-Flag-tagged nuclear extract. Activation of hydroxyl radical production by H2O2 is indicated. Shown is a Western blot probed with an anti-Flag antibody to monitor Gal11 cleavage. Asterisks indicate cleavage sites of Gal11 that are induced by FeBABE linked to either of the Gcn4 activation domains (black), the N-terminal activation domain only (blue), or the central activation domain only (red). (C) Summary of Gal11 cleavage sites generated by the Gcn4-FeBABE derivatives. The darkest vertical bars indicate the strongest cleavage. WT, wild type.
FIG. 5.
FIG. 5.
Multiple conserved elements contribute additively to Gal11 coactivator activity. (A) Gray boxes 1 to 5 indicate conserved motifs within Gal11. Elements 2 to 4 specifically bind Gcn4. Brackets indicate the positions of Glu-Asn- or Glu-Ala-rich regions flanking the activator-binding domains. (B) Strains containing wild-type (WT) Gcn4 and the indicated Gal11 mutations were induced with SM to induce starvation conditions before RNA was extracted and ARG3 mRNA was quantitated using RT-qPCR. SM cells were not induced. Blue, black, green, purple, and orange bars are colored according to the number of conserved Gal11 elements deleted. Experiments were performed in biological triplicate, and error bars represent the standard error of the mean. Brackets indicate the total number of conserved elements mutated in the N-terminal half of Gal11.
FIG. 6.
FIG. 6.
Characterization of Gal11 and Taf12 activator-binding domain mutants. (A) Western blotting of whole-cell yeast extracts from strains containing the indicated Gal11-HA derivatives (lanes 1 to 13) and Srb2-Flag. Cells were induced with SM as in other experiments. (B) Mediator (Srb2-Flag) was immunoprecipitated (IP) from whole-cell extracts made in panel A. Gal11-HA, Srb2-Flag, and Pgd1 levels were visualized by Western blotting. The values to the left of panel B are molecular sizes in kilodaltons. WT, wild type.
FIG. 7.
FIG. 7.
Multiple conserved Gal11 elements contribute to Gcn4 activation at genes with modest dependence on Gal11 coactivator function. (A to C) mRNA levels from the indicated Gcn4-dependent genes were determined using RNA from the experiment described in Fig. 5. Experiments were performed in biological triplicate, and error bars represent the standard error of the mean. WT, wild type.
FIG. 8.
FIG. 8.
Multiple conserved Gal11 elements contribute additively to Mediator recruitment. Strains containing wild-type (WT) Gcn4, epitope-tagged Srb2, and the indicated Gal11 derivatives were grown as described for Fig. 5 to 7, induced (except where noted otherwise), and cross-linked with formaldehyde, and cross-linking was analyzed using ChIP. ChIP assay to measure cross-linking of (A) Gal11-HA derivatives and (B) Srb2-Flag to the ARG3 promoter. Blue, gray, and white bars indicate GAL11, Δgal11, and strains lacking an epitope tag. Un, uninduced GAL11; no Tag, GAL11 strain lacking either an HA (A) or a Flag epitope (B). Black, green, purple, and orange bars are colored according to the number of conserved Gal11 elements deleted. Averages of biological duplicate experiments are expressed relative to the wild type, which was set at 1.0. Error bars indicate standard deviations.
FIG. 9.
FIG. 9.
Measurement of activator-Gal11 binding by FP. Assay for binding of conserved Gal11 domains to the indicated Oregon Green-labeled Gcn4 activation domains or the 6 × Ala Gcn4 derivative lacking transcription activation activity. Panels: A, Gal11 158-238; B, Gal11 430-680; C, Gal11 277-368; D, Gal11 6-90 (KIX domain). All assays were performed in triplicate, curves were fitted as described in Materials and Methods, and error bars represent standard deviations.
FIG. 10.
FIG. 10.
Summary of Gcn4 activation domain (AD) and Gal11-Gcn4 interactions. (A) Cross-linking targets of the tandem Gcn4 activation domains in PICs. (B) Three Gal11 activator-binding domains bind both Gcn4 activation domains. Gray boxes 1 to 5 indicate conserved regions within Gal11. Region 5 is required for association with the Mediator complex, and the KIX domain has an unknown function at Gcn4-regulated genes.
See this image and copyright information in PMC

References

    1. Altschuler, E. L., N. V. Hud, J. A. Mazrimas, and B. Rupp. 2000. Structure of polyglutamine. FEBS Lett. 472:166-168. - PubMed
    1. Barberis, A., J. Pearlberg, N. Simkovich, S. Farrell, P. Reinagel, C. Bamdad, G. Sigal, and M. Ptashne. 1995. Contact with a component of the polymerase II holoenzyme suffices for gene activation. Cell 81:359-368. - PubMed
    1. Bhaumik, S. R., T. Raha, D. P. Aiello, and M. R. Green. 2004. In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer. Genes Dev. 18:333-343. - "VSports在线直播" PMC - PubMed
    1. Bochkareva, E., L. Kaustov, A. Ayed, G. S. Yi, Y. Lu, A. Pineda-Lucena, J. C. Liao, A. L. Okorokov, J. Milner, C. H. Arrowsmith, and A. Bochkarev. 2005. Single-stranded DNA mimicry in the p53 transactivation domain interaction with replication protein A. Proc. Natl. Acad. Sci. U. S. A. 102:15412-15417. - PMC - PubMed
    1. Bourbon, H. M. 2008. Comparative genomics supports a deep evolutionary origin for the large, four-module transcriptional mediator complex. Nucleic Acids Res. 36:3993-4008. - "V体育安卓版" PMC - PubMed

"V体育官网入口" Publication types

V体育ios版 - MeSH terms

Substances (V体育官网入口)

LinkOut - more resources (VSports手机版)