Multiple regions of TBP participate in the response to transcriptional activators in vivo.

The TATA box-binding protein (TBP) plays an essential role in transcription by all three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP interacts with class-specific TBPassociated factors (TAFs) to form class-specific transcription initiation factors. For yeast Pol III transcription, TBP associates with Brf (from TFIIB-related factor) and B؆, two Pol III TAFs, to form Pol III transcription factor TFIIIB. Here, we identify TBP surface residues that are required for interaction with yeast Pol III TAFs. Ninety-one human TBP surface residue mutants with radical substitutions were analyzed for the ability to form stable gel shift complexes with purified Brf and B؆ and for their activities for in vitro synthesis of yeast U6 snRNA. Mutations in a large positively charged epitope extending from the top (that is, on the surface opposite the DNA-facing "saddle" of TBP) and onto the side of the first TBP repeat inhibited binding to Brf (residues K181, L185, R186, E206, R231, L232, R235, K236, R239, Q242, K243, K249, and F250). The TATA box-binding protein (TBP) plays an essential role in transcription by each of the three eukaryotic nuclear RNA polymerases, polymerases (Pol) I, II, and III. In each case, TBP associates with class-specific TBP-associated factors (TAFs) to form class-specific multisubunit transcription initiation factors that determine with which polymerase TBP functions (9,10,30). TBP also interacts with Pol II general transcription factors TFIIA and TFIIB (reviewed in references 2 and 27). Here, we sought to map the TBP surface that makes molecular contacts with the well-characterized yeast Pol III TAFs. We did this to better understand the architecture of the Pol III initiation complex, its relation to the Pol II initiation complex, and competing interactions between TBP and alternative TAFs and general transcription factors.In the yeast Saccharomyces cerevisiae, TBP associates with two Pol III TAFs, Brf (for TFIIB-related factor) and BЉ, to form the central Pol III initiation factor TFIIIB (7,14,40,42). Pol III transcribes small untranslated RNAs whose genes can be divided into three subclasses based on their promoter elements and their dependence on TFIIIA and TFIIIC (14,40,42). TFIIIB assembled from recombinant TBP, Brf, and BЉ is required for transcription of all three classes of Pol III genes where it is bound centered over a region about 30 bp upstream of the transcription start site (11). While the human homolog of BЉ has not been identified, TBP and Brf are conserved between yeast and human proteins (25,39), and the activity of a probable human homolog of BЉ has recently been described (37), suggesting that the entire TFIIIB assembly is conserved through evolution. Of the two yeast Pol III TAFs, TBP interacts more strongly with Brf to form a BЈ complex separable from the BЉ factor by ion-exchange chromatography (15,16). Incorporation of BЉ into a TBP-Brf-DNA complex stabilizes the DNA-protein complex to treatment with the polyanion heparin and to high salt ...

Section: Discussionmentioning

confidence: 59%

“…Moreover, human TBP with the triple alanine substitution of R231A ϩ R235A ϩ R239A showed reduced in vitro binding to Pol II TAF 250 (36). Consequently, we postulated that the Pol II activation epitope that nearly coincides with our proposed Brf interaction surface is an interaction surface for a Pol II TAF (2), as did Tansey et al (36).…”

Section: Discussionmentioning

confidence: 59%

Polymerase (Pol) III TATA Box-Binding Protein (TBP)-Associated Factor Brf Binds to a Surface on TBP Also Required for Activated Pol II Transcription

Shen

Kassavetis

Bryant

et al. 1998

“…Previous experiments have provided evidence for a specific role of TAF II 250 in vivo, as illustrated by a correlation between transcriptional activation by an altered-specificity TBP mutant and the ability of this mutant to bind TAF II 250 in vitro (56). Evidence for a role of TAF II 250 in transcriptional activation both in vitro and in vivo is also provided by experiments showing that stable expression of TAF II 250 can rescue a temperature-sensitive mutant hamster cell line containing a mutant TAF II 250 (61).…”

Section: Discussionmentioning

confidence: 99%

“…The functional relevance of this interaction was made evident from data showing that missense mutations in Sp1 that disrupt the interaction between TAF II 110 and Sp1 impair the ability of Sp1 to activate transcription (18). Whereas direct interactions between transactivators and Drosophila TAF II 230, or its human homolog TAF II 250, have not been observed, this TAF is essential for activated transcription in vitro (6,56,58,61). It has been speculated that TAF II 230/ TAF II 250 functions as a scaffold protein, binding directly to TBP and recruiting other TAFs to the TFIID complex (6,27,34,36,49,63).…”

mentioning

confidence: 97%

Functional Interaction between p53, the TATA-Binding Protein (TBP), and TBP-Associated Factors In Vivo

Farmer

Colgan

Nakatani

et al. 1996

102

The transcriptional activator p53 is known to interact with components of the general transcription factor TFIID in vitro. To examine the relevance of these associations to transcriptional activation in vivo, plasmids expressing a p53-GAL4 chimera and Drosophila TATA-binding protein (dTBP) were transfected into Drosophila Schneider cells. p53-GAL4 and dTBP displayed a markedly synergistic effect on activated transcription from a GAL4 site-containing reporter that was at least 10-fold greater than observed with other activators tested. A mutant p53 previously shown to be defective in both transcriptional activation in vivo and in binding to TBP-associated factors (TAFs) in vitro, although still capable of binding dTBP, did not cooperate with dTBP, suggesting that TAFs may contribute to this synergy. Providing further support for this possibility, transfected dTBP assembled into rapidly sedimenting complexes and could be immunoprecipitated with anti-TAF antibodies. While overexpression of any of several TAFs did not affect basal transcription, in either the presence or the absence of cotransfected dTBP, overexpression of TAF II 230 inhibited transcriptional activation mediated by p53-GAL4 as well as by GAL4-VP16 and Sp1. Overexpression of TAF II 40 and TAF II 60 also inhibited activation by p53-GAL4 but had negligible effects on activation by GAL4-VP16 and Sp1, while TAF II 110 did not affect any of the activators. TAF-mediated inhibition of activated transcription could be rescued by high levels of exogenous dTBP, which also restored full synergy. These data demonstrate for the first time that functional interactions can occur in vivo between TBP, TAFs, and p53.Exposure to DNA-damaging agents causes cellular levels of the p53 tumor suppressor protein to dramatically increase, resulting in a G 1 /S arrest in the cell cycle. This arrest is presumably due to the direct activation by p53 of genes involved in DNA repair, cell cycle regulation, and apoptosis (for reviews, see references 12 and 23). The role of p53 as a DNA-bindingdependent transcriptional activator is an important one since mutations that alter this ability lead to unregulated cellular growth and tumorigenesis (for a review, see reference 13). Therefore, it is of great interest to understand the mechanism of transcriptional activation by p53.Proper transcriptional activation of class II genes requires specific interactions between activators bound to regulatory elements and the general transcription factors that assemble on the TATA box and/or the initiator element (reviewed in reference 59). Besides TFIID, the factor that directly binds to the TATA box, the general factors include TFIIA, -B, -E, -F, and -H and RNA polymerase II (Pol II) (3, 11, 65), many of which have been shown to interact directly with transcriptional activators in vitro (8,31,40,46,64). The TATA-binding protein (TBP), the TFIID subunit that directly binds the TATA motif (for a review, see reference 26), has also been shown to interact directly with transcriptional activators in vit...

“…PCR fragments corresponding to aa 330 to 518, 238 to 518, and 238 to 572 were ligated into a modified pSBET vector (27) containing a glutathione S-transferase (GST)-coding sequence to generate constructs pSBET-GST-190RbRcRd, pSBET-GST-190RhRaRbRcRd, and pSBET-GST-190RhRaRbRcRdSer, respectively. GST fusion proteins were expressed in Escherichia coli BL21-DE3 (30), and lysates were prepared as described previously (31). Fusion proteins were bound to glutathione-agarose (Sigma), the beads were washed with phosphate-buffered saline containing 0.1% Nonidet P-40 and 10% glycerol, and the bound proteins were eluted with 10 mM glutathione in 50 mM Tris, pH 8.8.…”

Section: Methodsmentioning

confidence: 99%

The Large Subunit of Basal Transcription Factor SNAP_c Is a Myb Domain Protein That Interacts with Oct-1

Wong

Henry

et al. 1998

117

The human RNA polymerase II and III snRNA promoters have similar enhancers, the distal sequence elements (DSEs), and similar basal promoter elements, the proximal sequence elements (PSEs). The DSE, which contains an octamer motif, binds broadly expressed activator Oct-1. The PSE binds a multiprotein complex referred to as SNAP c or PTF. On DNAs containing both an octamer site and a PSE, Oct-1 and SNAP c bind cooperatively. SNAP c consists of at least four stably associated subunits, SNAP43, SNAP45, SNAP50, and SNAP190. None of the three small subunits, which have all been cloned, can bind to the PSE on their own. Here we report the isolation of cDNAs corresponding to the largest subunit of SNAP c , SNAP190. SNAP190 contains an unusual Myb DNA binding domain consisting of four complete repeats (Ra to Rd) and a half repeat (Rh). A truncated protein consisting of the last two SNAP190 Myb repeats, Rc and Rd, can bind to the PSE, suggesting that the SNAP190 Myb domain contributes to recognition of the PSE by the SNAP complex. SNAP190 is required for snRNA gene transcription by both RNA polymerases II and III and interacts with SNAP45. In addition, SNAP190 interacts with Oct-1. Together, these results suggest that the largest subunit of the SNAP complex is involved in direct recognition of the PSE and is a target for the Oct-1 activator. They also provide an example of a basal transcription factor containing a Myb DNA binding domain.The regulation of transcription initiation is mediated by the interplay between two classes of promoter elements: the basal promoter elements, which can be defined as those promoter elements sufficient to direct basal levels of transcription in vitro, and the regulatory elements, which modulate the levels of transcription. The basal elements are recognized by basal transcription factors, whereas the regulatory elements are recognized by either transcriptional activators or repressors. Eucaryotic activators are often modular, consisting of a DNA binding domain, which targets the activator to the correct promoter, and of activation domains, whose role is to enhance transcription (see references 21-23, 32, and 33 for reviews).The human snRNA gene family contains both RNA polymerase II and RNA polymerase III genes. The RNA polymerase II snRNA promoters consist of a proximal sequence element (PSE), which is sufficient to direct basal levels of transcription in vitro, and a distal sequence element, which activates basal transcription. The RNA polymerase III snRNA promoters are similar, except that basal transcription is directed by the combination of a PSE and a TATA box (reviewed in reference 9). The PSE is recognized by a multisubunit complex called the SNAP complex (SNAP c ) (7) or PTF (34). Since SNAP c can bind to the PSE on its own, it corresponds to a sequence-specific DNA binding basal transcription factor. SNAP c contains at least four subunits, SNAP43, SNAP45, SNAP50, and SNAP190, and cDNAs encoding the SNAP43 (7) or PTF ␥ (35), SNAP45 (24) or PTF ␦ (35), and SNAP50 (6) or PTF ␤ (2) su...