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. 2003 Oct 14;100(21):12003-8.
doi: 10.1073/pnas.2035253100. Epub 2003 Sep 23.

Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA

Greg T Cantin  1 , Jennitte L Stevens, Arnold J Berk
Affiliations

Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA

Greg T Cantin et al. Proc Natl Acad Sci U S A. .

Abstract

The interaction of activators with mediator has been proposed to stimulate the assembly of RNA polymerase II (Pol II) preinitiation complexes, but there have been few tests of this model. The finding that the major adenovirus E1A and mitogen-activated protein kinase-phosphorylated Elk1 activation domains bind to Sur2 uniquely among the metazoan mediator subunits and the development of transcriptionally active nuclear extracts from WT and sur2-/- embryonic stem cells, reported here, allowed a direct test of the model. We found that whereas VP16, E1A, and phosphorylated Elk1 activation domains each stimulate binding of mediator, Pol II, and general transcription factors to promoter DNA in extracts from WT cells, only VP16 stimulated their binding in extracts from sur2-/- cells. This stimulation of mediator, Pol II, and general transcription factor binding to promoter DNA correlated with transcriptional activation by these activators in WT and mutant extracts. Because the mutant mediator was active in reactions with the VP16 activation domain, the lack of activity in response to the E1A and Elk1 activation domains was not due to loss of a generalized mediator function, but rather the inability of the mutant mediator to be bound by E1A and Elk1. These results directly demonstrate that the interaction of activation domains with mediator stimulates preinitiation complex assembly on promoter DNA.

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Figures

Fig. 1.
Fig. 1.
Transcriptional activation by E1A and Elk1 in vitro requires the Sur2 mediator subunit. In vitro transcription was carried out by using a biotinylated linear DNA template immobilized on streptavidin-coated magnetic beads. (a) The immobilized template, G5TATA, contains five Gal4-binding sites upstream of a TATA-containing promoter driving transcription of a 233-bp G-less cassette. (b and c) Transcription was carried out on immobilized templates either without activator or with saturating amounts of Gal4 DNA-binding domain fused to the activation domain of VP16 (G4VP16), E1ACR3 (G4E1A), or the activation domain of Elk1 (G4Elk1). G4Elk1 bound to immobilized templates was either phosphorylated by the MAPK ERK2 or left unaltered. Nuclear extracts prepared from either WT or sur2–/– murine ES cells were used in the reactions, as indicated.
Fig. 2.
Fig. 2.
Requirement for the Sur2 mediator subunit in the E1A-induced binding of Pol II and GTFs to promoter DNA. Immobilized G5TATA DNA either without bound activator or with saturating amounts of recombinant Gal4-VP16 or -E1A was incubated with nuclear extract from WT or sur2–/– ES cells. Immobilized templates were then washed, and bound protein was eluted, resolved by SDS/PAGE, and analyzed by Western blotting using antibodies to the indicated proteins.
Fig. 3.
Fig. 3.
Requirement of the Sur2 mediator subunit for the Elk1-P-induced binding of Pol II and GTFs to promoter DNA. Nuclear extracts from WT and sur2–/– ES cells were incubated with the immobilized templates G5TATA or G-less (a DNA fragment lacking the five Gal4-binding sites and the core promoter). Immobilized G5TATA contained either no activator or saturating amounts of Gal4-VP16, -Elk1, or -Elk1 phosphorylated in vitro by recombinant ERK2 (G4Elk1-P). Binding was analyzed as in Fig. 2. Additional antibodies were used that recognize Elk1 and phosphorylated Elk1 (Elk1-P).
Fig. 4.
Fig. 4.
Stimulation of Pol II and GTF binding occurs in the absence of a canonical TATA box. (a)G5TATA and G5ΔTATA immobilized templates, with or without bound Gal4-VP16 or Gal4-E1A, were incubated with WT ES nuclear extract. Bound protein was washed, eluted, and subjected to SDS/PAGE and Western blotting as in Figs. 2 and 3. (b) RNase protection assay of in vitro transcripts transcribed from the strand complementary to the G-less cassette template strand. Transcription was from the G5TATA (Left) or G5ΔTATA (Right) templates in the presence (+) or absence of Gal4-VP16. The probe was transcribed by T7 RNA polymerase from the G5ΔTATA template cut with EcoRI. Arrows indicate RNase-protected fragments corresponding to transcripts with 5′ ends in the region of the Gal4 sites. Lines at the right indicate RNase-protected fragments corresponding to transcripts complementary to the full length of the probe. The positions of single-stranded DNA markers containing the indicated number of nucleotides are shown at the left.
Fig. 5.
Fig. 5.
Model of the known protein–protein and protein–DNA interactions during Gal4-E1A- and Gal4-Elk1-P-activated transcription. DNA binding by the Gal4-activation domain fusions and TBP to the template studied here was independent of the activation domain–Sur2 interaction. But all other interactions shown were stabilized by interactions of these activation domains with Sur2. DBD, DNA-binding domain; TAFs, TBP-associated factors; Med, mediator.
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