Multiple Layers of Cooperativity Regulate Enhanceosome-Responsive RNA Polymerase II Transcription Complex Assembly

Ellwood, Katharine; Huang, Wenliang; Johnson, Reid C.; Carey, Michael

doi:10.1128/mcb.19.4.2613

Cited by 44 publications

(46 citation statements)

References 71 publications

(81 reference statements)

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“…First, we determined total RNA content and the total poly(A) ϩ mRNA content of each of these strains. Total RNA was isolated from equal numbers of cells, and mRNA content was measured by slot blot hybridization with 32 P-labeled oligo(dT 20 ) as a probe (Fig. 3D).…”

Section: Deletion Of Region 4 From Taf1p Results In Decreased Steadysmentioning

confidence: 99%

“…(D) Slot blot hybridization of total RNA to measure poly(A) ϩ mRNA content. Serial threefold dilutions of RNA (10 to 0.33 g) were blotted and probed with end-labeled 32 P-oligo(dT 20 ). Slot blot analyses were done in triplicate, and intensities from 3.3 to 0.33 g of RNA spots were averaged; only a single analysis for each strain is shown.…”

Section: Deletion Of Region 4 From Taf1p Results In Decreased Steadysmentioning

confidence: 99%

“…It is possible that critical conformational changes in TFIID, required for proper functioning (17,20,22,28,30,32,50,53,71), are affected by the mutations in Taf1p-region 4 in the Ts47 mutant. Alternatively, despite the fact that these mutations did not result in total dissociation of the Tafps, they might have sufficiently weakened Tafp-Tafp associations such that the resulting complex displays functional defects.…”

Section: Discussionmentioning

confidence: 99%

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Molecular and Genetic Characterization of a Taf1p Domain Essential for Yeast TFIID Assembly

Singh

Bland

Weil

2004

Molecular and Cellular Biology

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Yeast Taf1p is an integral component of the multiprotein transcription factor TFIID. By using coimmunoprecipitation assays, coupled with a comprehensive set of deletion mutants encompassing the entire open reading frame of TAF1, we have discovered an essential role of a small portion of yeast Taf1p. This domain of Taf1p, termed region 4, consisting of amino acids 200 to 303, contributes critically to the assembly and stability of the 15-subunit TFIID holocomplex. Region 4 of Taf1p is mutationally sensitive, can assemble several Tafps into a partial TFIID complex, and interacts directly with Taf4p and Taf6p. Mutations in Taf1p-region 4 induce temperature-conditional growth of yeast cells. At the nonpermissive temperature these mutations have drastic effects on both TFIID integrity and mRNA synthesis. These data are consistent with the hypothesis that Taf1p subserves a critical scaffold function within the TFIID complex. The significance of these data with regard to TFIID structure and function is discussed.TFIID, one of several multiprotein general transcription factors required for RNA polymerase II (PolII)-catalyzed mRNA gene transcription, is comprised of the TATA-binding protein (TBP) and 14 distinct TBP-associated factors (TAFs or Tafps) that range in M r from 17,000 to 150,000 in Saccharomyces cerevisiae (62,63,65,75). These TFIID subunits share significant sequence conservation with their metazoan orthologs (1,22,26). In vitro, PolII needs six general transcription factors, TFIIA, -B, -D, -E, -F, and -H, to form functional preinitiation complexes (PICs) (12, 51, 54); TFIID plays several critical roles in this process. First, TFIID functions during promoter recognition by binding directly to the promoter (67,76,77). On promoters containing a TATA box, Tafps help stabilize TBP binding to the TATA element via interaction with TATA-proximal initiator DNA sequences (69). Tafp-DNA interactions may be particularly important for TFIID binding to promoters that lack a canonical TATA element and contain TATA-proximal initiator and distal promoter element sequences (13,15,42,69,76). Second, Tafps can function as coactivators during transcriptional activation by making direct contacts with specific activators leading to an increase in PolII PIC formation (1, 16). Finally, Taf1p contains several distinct enzymatic activities, those of histone acetyltransferase (HAT) (46), protein kinase (19), and ubiquitin ligase (55). These enzymatic activities presumably modify proteins that stimulate PIC formation and/or function, leading to PolII transcription initiation.In most contexts, the Tafp subunits of TFIID are essential for survival, as Tafp inactivation or depletion results in cessation of specific mRNA synthesis and loss of cell viability (3,4,33,34,56,59,64,65,78). Because of these critical roles, the composition, organization, assembly, structure, and function of the TFIID complex have been topics of great interest. TBP appears to be incorporated into the TFIID complex primarily through its interaction with the biparti...

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Section: Deletion Of Region 4 From Taf1p Results In Decreased Steadysmentioning

confidence: 99%

Section: Deletion Of Region 4 From Taf1p Results In Decreased Steadysmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Molecular and Genetic Characterization of a Taf1p Domain Essential for Yeast TFIID Assembly

Singh

Bland

Weil

2004

Molecular and Cellular Biology

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“…The first indication for this came from the work of Lieberman and Berk who demonstrated that not only do Zta and TBP interact directly, but the interaction stabilizes the association of TFIID with the TATA element (Lieberman & Berk, 1991). This was followed by studies describing the formation of stable initiation complexes containing Zta (Lieberman & Berk, 1991, 1994Chi & Carey, 1993Lieberman, 1994;Chi et al, 1995a, b;Ozer et al, 1996;Lieberman et al, 1997;Berk et al, 1998;Ellwood et al, 1999;Deng et al, 2001). Although it is inferred that the interaction between Zta and the general transcriptional machinery promotes transcription of ZRE-containing promoters, it remains an open question whether the interaction of Zta with the basic transcriptional machinery affects the expression of genes that do not contain a ZRE.…”

Section: Basic Transcriptional Machinerymentioning

confidence: 99%

bZIP proteins of human gammaherpesviruses

Sinclair

2003

Journal of General Virology

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“…PCR was employed to amplify a fragment from Ϫ990 to ϩ90 of the EBV BHLF-1 promoter using pBamW 2 YFSal G, which contains a segment of the B95-8 EBV genome spanning 40 to 61 kb (48). This fragment was subcloned into a mammalian CAT expression vector, generating HLCAT as previously described (13). A DNase I footprint between Z-3 and Z-4, from positions Ϫ116 to Ϫ128, was observed on this promoter when it was incubated with both ZEBRA and HMG-1.…”

Section: Methodsmentioning

confidence: 99%

Mechanism for Specificity by HMG-1 in Enhanceosome Assembly

Ellwood

Yen

Johnson

et al. 2000

Molecular and Cellular Biology

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Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.An emerging theme in eukaryotic gene expression is that promoter-and cell-specific transcription is achieved through regulated assembly of activators into nucleoprotein structures termed enhanceosomes (6,22,37). Enhanceosome assembly is mediated by cooperative protein-protein interactions dictated by the positioning of activator binding sites on a regulatory sequence and the concentration of relevant activators in a cell (6,43). Because interactions between activators generate energetically unfavorable DNA bends, architectural proteins that bend and twist the DNA are necessary to facilitate cooperative binding. An important issue in the field is how such flexure can be provided on a global level for the thousands of combinatorial activator arrays bound to genes in a eukaryotic nucleus (6,22,32,37,38,42).Both sequence-specific and nonspecific DNA architectural proteins have been identified. The largest family of eukaryotic architectural proteins contains the conserved HMG box, a 75-amino-acid sequence of known structure. Numerous examples exist of HMG-box proteins that bind DNA either specifically (e.g., LEF-1) or nonspecifically (e.g., HMG-1 and -2) (4). The function and mechanism of some sequence-specific architectural proteins have been established, while the nonspecific proteins have remained enigmatic. In this paper we examine how the abundant and relatively nonspecific HMG-1 and -2 proteins can function in a specific context.To provide a framework for the problem, consider the action of LEF-1 on the T-cell receptor alpha (TCR-␣) enhanceosome. LEF-1 or TCF-1 binds to an 8-bp sequence within the 75-bp TCR-␣ enhancer, bends the DNA, and stimulates cooperative binding of the flanking activators, PEBP2␣-Ets-1...

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Multiple Layers of Cooperativity Regulate Enhanceosome-Responsive RNA Polymerase II Transcription Complex Assembly

Cited by 44 publications

References 71 publications

Molecular and Genetic Characterization of a Taf1p Domain Essential for Yeast TFIID Assembly

Molecular and Genetic Characterization of a Taf1p Domain Essential for Yeast TFIID Assembly

bZIP proteins of human gammaherpesviruses

Mechanism for Specificity by HMG-1 in Enhanceosome Assembly

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