The general transcription factors IIA, IIB, IIF, and IIE are required for RNA polymerase II transcription from the human U1 small nuclear RNA promoter. (1/177)

RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.  (+info)

Phosphorylation of yeast TBP by protein kinase CK2 reduces its specific binding to DNA. (2/177)

Protein kinase CK2 is a ubiquitous Ser/Thr kinase which phosphorylates a large number of proteins including several transcription factors. Recombinant Xenopus laevis CK2 phosphorylates both recombinant Saccharomyces cerevisiae and Schizosaccharomyces pombe TATA binding protein (TBP). The phosphorylation of TBP by CK2 reduces its binding activity to the TATA box. CK2 copurifies with the transcription factor IID (TFIID) complex from HeLa cell extracts and phosphorylates several of the TBP-associated factors within TFIID. Taken together these findings argue for a role of CK2 in the control of transcription by RNA polymerase II through the modulation of the binding activity of TBP to the TATA box.  (+info)

Smubp-2 represses the Epstein-Barr virus lytic switch promoter. (3/177)

Smubp-2 is a novel transcription factor that was first identified through its interaction with the immunoglobulin Smu region (Mizuta et al., 1993) and has been cloned by virtue of its binding to two 12-O-tetradecanoylphorbol-13-acetate-responsive elements in the Epstein-Barr virus immediate-early BZLF1 promoter (Gulley et al., 1997). In this report, we examined the effect of Smubp-2 overexpression on BZLF1 prom oter activity. Overexpression of Smubp-2 in the B lymphocyte cell line BJAB caused repression of the BZLF1 gene promoter. A 14-bp region that partially overlaps with a 12-O-tetradecanoylphorbol-13-acetate-responsive element was required for maximal repression by Smubp-2, but some repression was also seen with a minimal promoter containing only the BZLF1 promoter TATA box and an initiation site. A 30-bp fragment containing the 14-bp region could transfer Smubp-2-mediated repression to heterologous promoters. Smubp-2 was found to associate with the basal transcription factor TATA binding protein (TBP) and to disrupt the formation of a stable TBP-TFIIA-DNA complex on the BZLF1 promoter TATA box and the adenovirus E1B promoter TATA box. Repression of the BZLF1 promoter by overexpressed Smubp-2 was rescued by overexpression of the basal factor TFIIA. These results suggest that complete repression of the BZLF1 promoter by Smubp-2 involves disruption of a functional TBP-TFIIA-TATA box complex and requires the -93 bp-to--79 bp region of the promoter.  (+info)

Mechanism of transcriptional repression of E2F by the retinoblastoma tumor suppressor protein. (4/177)

The retinoblastoma tumor suppressor protein (pRB) is a transcriptional repressor, critical for normal cell cycle progression. We have undertaken studies using a highly purified reconstituted in vitro transcription system to demonstrate how pRB can repress transcriptional activation mediated by the E2F transcription factor. Remarkably, E2F activation became resistant to pRB-mediated repression after the establishment of a partial (TFIIA/TFIID) preinitiation complex (PIC). DNase I footprinting studies suggest that E2F recruits TFIID to the promoter in a step that also requires TFIIA and confirm that recruitment of the PIC by E2F is blocked by pRB. These studies suggest a detailed mechanism by which E2F activates and pRB represses transcription without the requirement of histone-modifying enzymes.  (+info)

Multiple layers of cooperativity regulate enhanceosome-responsive RNA polymerase II transcription complex assembly. (5/177)

Two coordinate forms of transcriptional synergy mediate eukaryotic gene regulation: the greater-than-additive transcriptional response to multiple promoter-bound activators, and the sigmoidal response to increasing activator concentration. The mechanism underlying the sigmoidal response has not been elucidated but is almost certainly founded on the cooperative binding of activators and the general machinery to DNA. Here we explore that mechanism by using highly purified transcription factor preparations and a strong Epstein-Barr virus promoter, BHLF-1, regulated by the virally encoded activator ZEBRA. We demonstrate that two layers of cooperative binding govern transcription complex assembly. First, the architectural proteins HMG-1 and -2 mediate cooperative formation of an enhanceosome containing ZEBRA and cellular Sp1. This enhanceosome then recruits transcription factor IIA (TFIIA) and TFIID to the promoter to form the DA complex. The DA complex, however, stimulates assembly of the enhanceosome itself such that the entire reaction can occur in a highly concerted manner. The data reveal the importance of reciprocal cooperative interactions among activators and the general machinery in eukaryotic gene regulation.  (+info)

Phosphorylation of TFIIA stimulates TATA binding protein-TATA interaction and contributes to maximal transcription and viability in yeast. (6/177)

Posttranslational modification of general transcription factors may be an important mechanism for global gene regulation. The general transcription factor IIA (TFIIA) binds to the TATA binding protein (TBP) and is essential for high-level transcription mediated by various activators. Modulation of the TFIIA-TBP interaction is a likely target of transcriptional regulation. We report here that Toa1, the large subunit of yeast TFIIA, is phosphorylated in vivo and that this phosphorylation stabilizes the TFIIA-TBP-DNA complex and is required for high-level transcription. Alanine substitution of serine residues 220, 225, and 232 completely eliminated in vivo phosphorylation of Toa1, although no single amino acid substitution of these serine residues eliminated phosphorylation in vivo. Phosphorylated TFIIA was 30-fold more efficient in forming a stable complex with TBP and TATA DNA. Dephosphorylation of yeast-derived TFIIA reduced DNA binding activity, and recombinant TFIIA could be stimulated by in vitro phosphorylation with casein kinase II. Yeast strains expressing the toa1 S220/225/232A showed reduced high-level transcriptional activity at the URA1, URA3, and HIS3 promoters but were viable. However, S220/225/232A was synthetically lethal when combined with an alanine substitution mutation at W285, which disrupts the TFIIA-TBP interface. Phosphorylation of TFIIA could therefore be an important mechanism of transcription modulation, since it stimulates TFIIA-TBP association, enhances high-level transcription, and contributes to yeast viability.  (+info)

Corepressor required for adenovirus E1B 55,000-molecular-weight protein repression of basal transcription. (7/177)

Adenovirus E1B 55,000-molecular-weight protein (55K) binds to host cell p53, stabilizing it, greatly increasing its affinity for its cognate DNA-binding site, and converting it from a regulated activator to a constitutive repressor. Here we analyzed the mechanism of repression by the p53-E1B 55K complex. E1B 55K repression requires that 55K be tethered to the promoter by binding directly to DNA-bound p53. Transcription from an assembled, p53-activated preinitiation complex was not repressed by the subsequent addition of E1B 55K, suggesting that either sites of 55K interaction with p53 or targets of 55K in the preinitiation complex are blocked. Specific E1B 55K repression was observed in reactions lacking TFIIA and with recombinant TATA-binding protein in place of TFIID, conditions under which p53 does not activate transcription. Thus, E1B 55K does not simply inhibit a p53-specific activation mechanism but rather blocks basal transcription. As a consequence, E1B 55K may repress transcription from any promoter with an associated p53-binding site, no matter what other activators associate with the promoter. E1B 55K did not repress basal transcription in reactions with recombinant and highly purified general transcription factors and RNA polymerase II but rather required a corepressor that copurifies with the polymerase.  (+info)

Identification of a general transcription factor TFIIAalpha/beta homolog selectively expressed in testis. (8/177)

In this paper we describe the isolation of a cDNA that encodes a human TFIIAalpha/beta-like factor (ALF). The open reading frame of ALF predicts a protein of 478 amino acids that contains characteristic N- and C-terminal conserved domains separated by an internal nonconserved domain. In addition, a rare ALF-containing cDNA, which possesses an extended N terminus (Stoned B/TFIIAalpha/beta-like factor; SALF) has also been identified. The results of Northern and dot blot analyses show that ALF is expressed almost exclusively in testis; in contrast, TFIIAalpha/beta and TFIIAgamma are enriched in testis but are also widely expressed in other human tissues. Recombinant ALF (69 kDa) and TFIIAgamma (12 kDa) polypeptides produced in Escherichia coli form an ALF/gamma complex that can stabilize TBP-TATA interactions in an electrophoretic mobility shift assay. The ALF/gamma complex is also able to restore transcription from the adenovirus major late promoter using HeLa cell nuclear extracts that have been depleted of TFIIA. Overall, the data show that ALF is a functional homolog of human general transcription factor TFIIAalpha/beta that may be uniquely important to testis biology.  (+info)