BIOLOGICAL OVERVIEW

The gene Tbp-related factor (Trf) codes for cell-type specific protein related to TATA-binding protein (Tbp), which directs transcription by cellular RNA polymerases. Eukaryotic nuclear and archaeal transcription apparatuses share an essential common feature: the RNA polymerase (pol) is brought to the transcriptional start site by a DNA-bound assembly of transcription initiation proteins. The promoter-marking assembly of archaeal RNA polymerases and the core promoter-marking assembly of eukaryotic pol II each consist of two proteins: TATA-binding protein (TBP) and the phylogenetically related transcription factor B (TFB) or TFIIB (for archaeal or pol II transcription systems, respectively) (Kassavetis, 1998 and references therein).

It has been widely accepted that TBP is a universal factor that plays a central role in eukaryotic transcription by all three RNA polymerases (I, II, and III). Indeed, TBP is highly conserved among all the eukaryotes studied and its universal involvement in all three RNA polymerases has been established for human and yeast. Contrary to this general expectation, Drosophila TBP may not be a major player in directing RNA pol III transcription. Instead, the TBP-related factor Trf was found to be a principal participant in Drosophila pol III transcription. In vitro transcription studies with multiple classes of pol III templates failed to show any significant contribution of TBP, but instead revealed a clear contribution by Trf (Takada, 2000).

The promoter-marking assembly of pol III differs slighly from that of polII. TFIIIB, is composed of three subunits: TBP, Brf (named for its relatedness to TFIIB), and B". In Saccharomyces cerevisiae, the three components of TFIIIB are held together by a more stable interaction between Brf and TBP (which together make up the B' component of TFIIIB) and by a weaker interaction between TBP-DNA-bound Brf and B"; a weaker direct B"-TBP interaction has also been noted. Each of these TFIIIB components is required for all pol III transcription in yeast. Homologs of Brf and B" serve as components of the pol III system of humans but are not ubiquitously required. It appears instead that specialized alternative promoter-marking assemblies participate in different classes of human pol III promoters (Kassavetis, 1998 and references therein).

The homology relationship of Brf with TFIIB is confined to the N-terminal half of the much larger Brf; similarities between the C-proximal half of Brf and proteins of the pol II transcription initiation apparatus have not been discerned, at least at the level of the primary amino acid sequence. Drosophila BRF was isolated as a TRF1-associated factor, while no significant amount of TBP:BRF complex was observed. Immunodepletion of Drosophila nuclear extracts followed by reconstitution with purified recombinant TRF1:BRF directly implicated this complex in mediating RNA pol III transcription. Thus, it appears that in Drosophila TRF1:BRF contributes significantly to RNA pol III transcription initiation in place of a TBP:BRF complex (Takada, 2000).

Before describing the biology of Trf, a short review of Tbp and the protein complex TFIID will be given. TFIID is multiprotein complex containing the TATA box binding protein (Tbp). In Drosophila, at least seven other proteins are known as TAFs or Tbp associated factors. The first protein recruited to the promoter is Tbp, which serves to induce a bend in the DNA. The 240 kD subunit of TFIID (Tbp-associated factor 250kD) contains an HMG-box, bromodomains, a serine kinase, and histone acetyltransferase activity. The smaller subunits are similar in structure to histones. Drosophila Tbp-associated factor 60kD (also known as dTAFII62) and Tbp-associated factor 40kD (also know as dTAFII42) are homologous to human hTAFII80 and hTAFII31 respectively; Drosophila and human proteins are homologous to histone H3 and histone H4, respectively. Both Drosophila and human TFIID also contain dTAFII30 alpha and hTAFII20, two putatitive histone H2B homologs. In solution and in the crystalline state, the dTAFII42/dTAFII62 complex exists as a heterotetramer, resembling the (H3/H4)2 heterotetrameric core of the histone octamer, suggesting that TFIID contains a histone octamer-like substructure. Tbp participates in TFIID function even in promoters lacking a TATA box (Xie, 1996 and references).

Considerable information is available about Drosophila Tbp and its interaction with Tbp-associated factor 250kD, the largest subunit of TFIID. The 80-residue N-terminal region of Drosophila TAF(II)250 can bind directly to Tbp and inhibit its function. This N-terminal region binds to the concave surface of Tbp, which is important for TATA box binding. A point mutation (L114K) on this concave surface destroys the ability of Tbp to bind transcriptional activator VP16 and to mediate VP16-dependent activation in vitro, but has no effect on basal transcription. The same Tbp mutation eliminates Tbp binding to the N-terminal region of TAF(II)250. Consistent with these L114K mutation effects, the N-terminal region of TAF(II)250 and the VP16 activation domain compete for binding to wild-type Tbp. These results indicate that transcriptional regulation may involve, in part, competitive interactions between transcriptional activators and TAFs on the Tbp surface (Nishikawa, 1997).

Several transcription factors have been shown to interact with Drosophila Tbp. Three are discussed here: (1) There is a direct interaction between Even-skipped and the TATA-binding protein. EVE can efficiently either block or squelch promoters whose activity can be enhanced by coexpression of Tbp. Squelching does not require Eve DNA-binding sites but is dependent on the presence of the Eve repression domain. Blocking requires an intact Eve repression domain and the conserved C-terminus of Tbp. The Eve homeodomain is also required for these associations, suggesting that the homeodomain itself may function in protein-protein interactions (Um, 1995).

(2)The Dorsal Switch Protein (DSP1), a transcriptional corepressor that serves to convert Dorsal into a transcriptional repressor, binds directly to the TATA binding protein (Tbp), and forms a stable ternary complex with Tbp bound to DNA. DSP1 preferentially disrupts the DNA binding of Tbp complexes containing TFIIA, a general transcription factor implicated in activated transcription pathways. DSP1 inhibits activated (but not basal) transcription reactions in vitro. The ability of DSP1, both to interact with Tbp and to repress transcription, maps to the carboxy-terminal domain of DSP1, which contains two HMG boxes (Kirov, 1996).

(3) Drosophila Tbp interaction with transcription factors also involves a coactivator of the transcription factor FTZ-F1. The coactivator, Multiprotein bridging factor 1 (MBF1), makes possible a connection, or bridges, the TATA box-binding protein (Tbp) and the nuclear hormone receptor FTZ-F1. MBF1 is functional in interactions with Tbp and a positive cofactor MBF2. MBF1 makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the binding of FTZ-F1 to its recognition site. The central region of MBF1 (residues 35-113) is essential for the binding of FTZ-F1, MBF2, and Tbp. MBF1, in the presence of MBF2, and FTZ622 bearing the FTZ-F1 DNA-binding domain, support selective transcriptional activation of the fushi tarazu gene. Mutations that disrupt the binding of FTZ622 to DNA or MBF1, or an MBF2 mutation that disrupts the binding to MBF1, all abolish the selective activation of transcription. These results suggest that tethering the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription (Takemaru, 1997).

The interation of Tbp with transcription factors points to the importance of Tbp in transcriptional regulation and suggests that an understanding of these interactions will serve to elucidate the complexity and specificity of gene regulation. The discovery of a tissue specific TATA-binding protein in Drosophila adds another dimension to the Tbp story and suggests that a similar complexity may exist in higher vertebrates. TBP-related factor (Trf) was discovered in a genetic screen for mutant flies that show leg shaking under ether anesthesia, a phenotype that is attributed to defects in nervous system function. In fact, Trf is expressed specifically in the brain lobe and ventral cord as well as in the gonads (Hansen, 1997).

Trf can substitute for Tbp in directing initiation by RNA polymerase II; Trf has a similar general transcription factor (GTF) requirement as that observed for Tbp. A complete transcription reaction mixture including Trf, TFIIA, TFIIB, TFIIE, TFIIF, TFIIH and RNA polymerase II was prepared. Omission of TFIIA results in a modest decrease in activity. By contrast, transcription is abolished by removal of TFIIB, TFIIE, TFIIF, or RNA polymerase II, while removal of TFIIH results in a residual low level of activity. These results confirm that Trf has a similar GTF requirement as that observed for Tbp. Thus Trf can substitute for Tbp in order to direct transcription initiation by RNA polymerase II; according to several criteria, Trf appears to be a functional homolog of Tbp (Hansen, 1997).

Trf was found to be present in a large protein complex that proved to be difficult to purify because of its low concentration. Even when starting with more than 3 kg of embryos, the Trf complex could not be purified in sufficient quantities for a comprehensive biochemical analysis. As an alternative, Trf was isolated from cultured Drosophila cells known as Schneider cells. Surprisingly, the Trf complex does not contain the ubiquitous TAF subunits found associated with Tbp. The core subunit of TFIID is absent: Tbp-associated factor 250kD. Also absent are each of the seven other TAFs that make up TFIID. It is speculated that, like the ubiquitous TAFs, neural TAFs may help direct direct Trf to a specific promoter and mediate activation by a select subset of enhancer binding factors (Hansen, 1997).

Trf is present as a constituent of larval salivary gland polytene chromosomes, revealing information about potential Trf target gene selectivity. Trf is found in 17 out of up to 600 resolvable sites, suggesting that Trf is localized in a highly gene-specific pattern at the same time that Tbp is present almost ubiquitously. The identified sites have neural or gonadal functions. For example, 63AB contains the shaker cognate b (shab) gene that encodes a potassium channel. This finding is consistent with the observation that trf mutant flies display a leg shaking phenotype, generally attributed to abnormalities in potassium channel function. Trf also associates with the location of quiver, which is also associated with a shaker phenotype. The genes maleless/no action potential exhibit Trf association as well as a number of chromosomal sites that contain one or more tRNA genes. These data suggests that Trf, like Tbp, may also play a role in RNA polymerase III transcription, at least in the salivary gland (Hansen, 1997).

GENE STRUCTURE

Bases in 5' UTR - 75

Bases in 3' UTR - 116

PROTEIN STRUCTURE

Amino Acids - 252

Structural Domains

Trf, a Drosophila gene product, has similarity to Tbp and produces a highly restricted expression pattern in the embryo. This TBP-related factor is a DNA-binding protein and suggests that TBP-related factor is a sequence-specific transcription factor that shares the DNA-binding properties of Tbp (Crowley, 1993).

date revised: 10 November 97

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