The Drosophila sequence-specific DNA binding protein, Adf-1, is capable of activating transcription of the alcohol dehydrogenase gene, Adh, and is implicated in the transcriptional control of other developmentally regulated genes. We have cloned the cDNA encoding Adf-1 by generating specific DNA probes deduced from partial amino acid sequence of the protein. Several cDNA clones encoding an extended open reading frame were isolated from a phage lambda library. The complete amino acid sequence of Adf-1 deduced from the longest cDNA reveals structural similarities to the putative helix-turn-helix DNA binding motif of Myb and Myb-related proteins. DNA sequence analysis of genomic clones and Northern blot analysis of mRNA suggest that Adf-1 is a single-copy gene encoding a 1.9-kb transcript. Purified recombinant Adf-1 expressed in Escherichia coli binds specifically to Adf-1 recognition sites and activates transcription of a synthetic Adh promoter in vitro in a manner indistinguishable from the protein purified from Drosophila. Temporally staged Drosophila embryos immunochemically stained with affinity-purified anti-Adf-1 antibodies indicate that Adf-1 protein is not detectable in very early embryos and does not appear to be maternally inherited. During later stages of embryogenesis, Adf-1 appears to be expressed in the nucleus of most somatic cells in the embryo with possibly higher concentrations found in some tissues.

The basal factor TFIIA has been shown to act early during initiation in both the mammalian and yeast transcription systems, but a TFIIA-like activity has not been identified in Drosophila. While characterizing the Drosophila TFIID complex, we discovered that a 30-kD protein that cofractionated with dTFIID was homologous to the previously identified, large subunit of yeast TFIIA. Here, we report the cloning and biochemical characterization of Drosophila TFIIA-L. Coimmunoprecipitation studies with anti-dTBP, anti-dTFIIA-L, and anti-TAF antibodies indicated a tight association of the endogenous dTFIIA and dTFIID. However, dTFIIA could be dissociated from dTFIID under conditions that did not elute the TAFs, and the eluted material had mobility shift and transcriptional activities associated with TFIIA. Peptide sequence and Western analysis with antibodies raised against the amino- and carboxy-terminal portions of recombinant dTFIIA-L revealed that a precursor 48-kD species was cleaved in vivo, giving rise to the 30- and 20-kD subunits of dTFIIA that remain associated with each other and with dTFIID. Protein-protein interaction assays identified dTBP and dTAFII110 as targets for binding TFIIA in the TFIID complex. These results suggest that TFIIA may form a specific complex with both TAFs and other components of the transcriptional machinery during formation of the initiation complex.

The multisubunit transcription factor TFIID is an essential component of the RNA polymerase II initiation apparatus. Recent studies suggest that TFIID subunits, or TAFs associated with the TATA-binding protein (TBP), play a critical role in modulating transcriptional activation by sequence-specific DNA-binding factors. Thus far, six of the largest TAFs associated with Drosophila TFIID have been cloned and partially characterized. Here, we report the molecular cloning, expression, and subunit interaction specificities of two small molecular mass TAFs. Both dTAFII30 alpha and dTAFII30 beta are associated with TFIID via interactions with other TAFs, including dTAFII250, dTAFII150, and dTAFII110. In addition, dTAFII30 alpha also contacts dTBP. The carboxy-terminal half of dTAFII110 was found to contact a short 67-amino-acid region of dTAFII30 alpha, which is predicted to form two potential alpha-helices, one of which is amphipathic. Interestingly, dTAFII30 alpha also appears to multimerize through its carboxy-terminal region. Although neither dTAFII30 alpha nor dTAFII30 beta have been found to interact with specific activators thus far, it is intriguing that both bind other TAFs such as dTAFII110 and dTAFII150, which are the targets of activation domains. Our studies suggest that both of the small subunits of TFIID play a role in the assembly of the complex and may contribute to the stability of multiple TAF-TAF interactions.

We show that the activities of two Ets-related transcription factors required for normal eye development in Drosophila, pointed and yan, are regulated by the Ras1/MAPK pathway. The pointed gene codes for two related proteins, and we show that one form is a constitutive activator of transcription, while the activity of the other form is stimulated by the Ras1/MAPK pathway. Mutation of the single consensus MAPK phosphorylation site in the second form abrogates this responsiveness. yan is a negative regulator of photoreceptor determination, and genetic data suggest that it acts as an antagonist of Ras1. We demonstrate that yan can repress transcription and that this repression activity is negatively regulated by the Ras1/MAPK signal, most likely through direct phosphorylation of yan by MAPK.

The general transcription factor TFIID is a multiprotein complex containing the TATA-binding protein and several associated factors (TAFs), some of which may function as coactivators that are essential for activated, but not basal, transcription. Here we describe the isolation and characterization of the first gene encoding a TAF protein. The deduced amino acid sequence of TAF110 revealed the presence of several glutamine- and serine/threonine-rich regions reminiscent of the protein-protein interaction domains of the regulatory transcription factor Sp1 that are involved in transcription activation and multimerization. In both Drosophila cells and yeast, TAF110 specifically interacts with the glutamine-rich activation domains of Sp1. Moreover, purified Sp1 selectively binds recombinant TAF110 in vitro. These findings taken together suggest that TAF110 may function as a coactivator by serving as a site of protein-protein contact between activators like Sp1 and the TFIID complex.

In Drosophila and human cells, the TATA binding protein (TBP) of the transcription factor IID (TFIID) complex is tightly associated with multiple subunits termed TBP-associated factors (TAFs) that are essential for mediating regulation of RNA polymerase II transcription. The Drosophila TAFII150 has now been molecularly cloned and biochemically characterized. The deduced primary amino acid sequence of dTAFII150 reveals a striking similarity to the essential yeast gene, TSM-1. Furthermore, like dTAFII150, the TSM-1 protein is found associated with the TBP in vivo, thus identifying the first yeast homolog of a TAF associated with TFIID. Both the product of TSM-1 and dTAFII150 bind directly to TBP and dTAFII250, demonstrating a functional similarity between human and yeast TAFs. Surprisingly, DNA binding studies indicate that purified recombinant dTAFII150 binds specifically to DNA sequences overlapping the start site of transcription. The data demonstrate that at least one of the TAFs is a sequence-specific DNA binding protein and that dTAFII150 together with TBP are responsible for TFIID interactions with an extended region of the core promoter.