Binding of general transcription factor TFIIB to an acidic activating region

Abstract

A central issue in eukaryotic transcriptional regulation is the mechanism by which promoter-specific transcription factors (activators) stimulate transcription. Two lines of evidence indicate that the general transcription factor TFIIB is a pivotal component in the mechanism by which an acidic activator functions. First, during assembly of the preinitiation complex TFIIB binding is a rate-limiting step enhanced by an acidic activator. Second, the TFIIB activity in a HeLa cell nuclear extract is specifically retained on a column containing an acidic activating region. But because our previous study monitored only TFIIB activity, it remains possible that the interaction between TFIIB and the acidic activating region is mediated through additional proteins, for example, those designated as adaptors, coactivators or mediators. A complementary clone encoding TFIIB has recently been isolated and shown to encode a polypeptide of relative molecular mass 35,000. Here we report that TFIIB expressed in and purified from Escherichia coli (recombinant TFIIB) binds directly to the potent acidic activating region of the herpes simplex virus-1 VP16 protein.

Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

“…The effects of TFIIB mutations on activation by GAL4-VP16 in vitro (90) suggest that an activator-TFIIB interaction is also important for transactivation. One experiment with the F442P mutation in VP16 led to the same conclusion (66). In other studies, neither the F442P mutation in the aminoterminal portion of the VP16 activation domain nor mutations in important phenylalanine residues in the carboxy-terminal portion of the VP16 activation domain, which all strongly reduce transactivation in vivo (17,88,111), affected the binding to VP16 of TFIIB (30,34,111).…”

“…To determine in a systematic way which general initiation factors bind to VP16, GST-VP16 fusion proteins were produced in Escherichia coli and used as ligands for affinity chromatography (66). The affinity columns were loaded with HeLa whole-cell extracts, and bound proteins were eluted with salt and assayed for the general factors required for initiation by RNA polymerase II in vitro at the adenovirus major late promoter.…”

“…2b, lane 7). However, mutations at Phe-473 and Phe-475, when combined with a mutation at the amino-terminal 442 position, significantly reduced transactivation by VP16 (88) (30,103), TFIIB (66), and DNA replication factor A (40, 63), it was possible that one of these proteins acts as a bridge between VP16 and TFIIH. To test this possibility, the TFIIH activity eluted from a GST-VP16 column was further purified by phosphocellulose chromatography (Fig.…”

“…These include activation domains in VP16 (30,66), Epstein-Barr virus R (70), and human c-Rel (118) and CTF (57), as well as COUP-TF and other members of the nuclear receptor superfamily (1,49 (112,114). Other experiments indicate that a direct interaction of the activator with TBP and/or TFIIB leads to recruitment of TFIIB into the preinitiation complex (11,57,65,66,105). When the DNA template contains multiple activator-binding sites, acidic activators also stimulate a subsequent step in the assembly of the preinitiation complex involving recruitment of the other general initiation factors and RNA polymerase II (11).…”

Oncogenic EWS-Fli1 interacts with hsRPB7, a subunit of human RNA polymerase II

“…These data indicate that additional interactions with components of the transcription machinery or transcription factors might be necessary for the high transactivation potential of EWS-Fli1. We know from virion protein VP16 that a single strongly activating domain can contact four di erent transcription factors (Xiao et al, 1994;Stringer et al, 1990;Lin et al, 1991;Goodrich et al, 1993). Since the EWS amino terminus is largely composed of a degenerate repeated peptide motif we cannot exclude that there are several interfaces contacting hsRPB7 downstream of the ®rst 82 amino acids.…”