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R Tjian

Publications and source records attributed to R Tjian.

At least 91 records · Page 5Linked to original sources
PubMed1993

A new factor related to TATA-binding protein has highly restricted expression patterns in Drosophila.

The TATA-binding protein TBP is necessary for the transcription of eukaryotic genes. Multi-protein complexes formed by TBP and different TBP-associated factors are involved in the initiation of transcription by polymerases I and II, and probably III as well. During the formation of an active initiation complex, TBP makes specific contacts with other proteins, for example TFIIB and RNA polymerase II (refs 2-4). Here we describe the cloning and characterization of a Drosophila gene product with considerable sequence similarity to TBP and a highly restricted expression pattern in the embryo. This TBP-related factor is a DNA-binding protein but is not likely to be a basal transcription factor. Our results suggest that TBP-related factor is a sequence-specific transcription factor that shares the DNA-binding properties of TBP.

Animals↗
PubMed1993

Molecular cloning and functional analysis of Drosophila TAF110 reveal properties expected of coactivators.

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.

Amino Acid Sequence↗
PubMed1993

Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex.

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.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and characterization of dTAFII30 alpha and dTAFII30 beta: two small subunits of Drosophila TFIID.

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.

Amino Acid Sequence↗
PubMed1993

Drosophila tissue-specific transcription factor NTF-1 contains a novel isoleucine-rich activation motif.

The Drosophila tissue-specific transcription factor NTF-1 provides a useful model system for studying the mechanisms by which promoter-selective factors control the development of a multicellular organism. A number of promoters that may be targets of NTF-1 regulation have been identified. For example, NTF-1 plays a critical role in the tissue-specific expression of the Drosophila Dopa decarboxylase gene. Additionally, by using in vitro assays, it has been possible to characterize the mechanism of NTF-1 activation, revealing its dependence on specific coactivators, or TAFs. Here, we report the use of both in vivo and in vitro assays to identify the functional domains of NTF-1. These consist of an unusually large, unique DNA-binding and dimerization domain, as well as a novel, isoleucine-rich activation domain. This 56-amino-acid activation region fails to interact with the putative Sp1 coactivator, dTAFII110, and thus appears to use a mechanism distinct from the glutamine-rich activation domain of Sp1. Additionally, NTF-1 appears to activate transcription in a species-specific manner, utilizing distinct domains in Drosophila and yeast.

Amino Acid Sequence↗
PubMed1992

The TATA-binding protein and associated factors are integral components of the RNA polymerase I transcription factor, SL1.

We have previously shown that the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs) are required for regulated transcriptional initiation by RNA polymerase II. Here we report the biochemical properties of the RNA polymerase I promoter selectivity factor, SL1, and its relationship to TBP. Column chromatography and glycerol gradient sedimentation indicate that a subpopulation of TBP copurifies with SL1 activity. Antibodies directed against TBP efficiently deplete SL1 transcriptional activity, which can be restored with the SL1 fraction but not purified TBP. Thus, TBP is necessary but not sufficient to complement SL1 activity. Analysis of purified SL1 reveals a complex containing TBP and three distinct TAFs. Purified TAFs reconstituted with recombinant TBP complement SL1 activity, and this demonstrates that TBP plus novel associated factors are integral components of SL1. These findings suggest that TBP may be a universal transcription factor and that the TBP-TAF arrangement provides a unifying mechanism for promoter recognition in animal cells.

Antibodies↗
PubMed1992

Cloning of Drosophila transcription factor Adf-1 reveals homology to Myb oncoproteins.

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.

Alcohol Dehydrogenase↗
PubMed1992

Eukaryotic coactivators associated with the TATA box binding protein.

Recent studies of regulated RNA polymerase II transcription have uncovered a new class of molecules called coactivators. These are tightly associated with the TATA box binding protein and are required in addition to promoter-specific activators and the basal transcription factors in order to achieve stimulated levels of transcription.

Animals↗
PubMed1992

Multiple domains of the RNA polymerase I activator hUBF interact with the TATA-binding protein complex hSL1 to mediate transcription.

Recent evidence suggests that transcription initiation by all three eukaryotic RNA polymerases involves a complex of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). Here, we map the functional domains of the nucleolar HMG box protein hUBF, which binds to the human rRNA promoter and stimulates transcription by RNA polymerase I through cooperative interactions with a distinct TBP-TAF complex, hSL1. DNase I footprint analysis of mutant hUBF proteins and of a synthetic peptide of 84 amino acids reveals that HMG box 1 is necessary and sufficient for DNA sequence specificity, whereas other HMG boxes and the amino terminus modulate the binding efficiency. hUBF contains multiple activation domains that include the acidic carboxyl terminus and three HMG boxes. HMG boxes 3 and 4 and the acidic tail contribute significantly to an extended footprinting pattern in the presence of hSL1, suggestive of specific protein-protein interactions. Moreover, the inability of xUBF from Xenopus laevis to form an initiation complex with hSL1 can be overcome by hybrid proteins containing human HMG box 4 and the acidic carboxyl terminus. These results strongly suggest an important role of transcription activation domains of hUBF in mediating interactions with the TBP-TAF complex hSL1.

Animals↗
PubMed1992

The cell-type-specific activator region of c-Jun juxtaposes constitutive and negatively regulated domains.

Dissection of the cell-type-specific activation region in c-Jun reveals two functionally separable regulatory subdomains. One subdomain (a1) functions as a transcriptional activator; adjacent to it is a newly identified domain (epsilon) which, together with the previously defined delta region, interacts with a cellular factor that modulates the action of a1. Mutants that lack epsilon are constitutively active and do not interact with the cell-type-specific repressor, whereas mutants that have sustained changes in a1 exhibit a reduced trans-activation potential but retain the ability to interact with the repressor. This bipartite and modular organization of the a1/epsilon domain is further established by demonstrating that a1 can be replaced by the heterologous acidic activator of VP16 and retain proper negative regulation by the cell-specific c-Jun inhibitor along with epsilon and delta. Repression of Jun activity by the inhibitor is not caused by a change in stability, nuclear localization, or DNA-binding activity of the protein. Instead the inhibitor apparently regulates transcriptional activation by interacting directly with delta/epsilon and perhaps masking the a1 domain. These studies suggest that multifunctional activation domains, which are structurally complex, may play an important role in the mechanisms that govern inducible tissue-specific gene expression.

Amino Acid Sequence↗
PubMed1991

Structure and functional properties of human general transcription factor IIE.

The general transcription factor IIE (TFIIE) is an essential component of the eukaryotic RNA polymerase II initiation complex. We have isolated human complementary DNA clones for both the subunits of TFIIE. Using purified recombinant proteins we find that both subunits are essential to form a stable preinitiation complex and to reconstitute basal-level and Sp1-activated transcription in vitro. Analysis of their predicted amino-acid sequences reveals several intriguing structural motifs that could provide insight into the role of TFIIE in transcription initiation.

Adenosine Triphosphate↗
PubMed1991

Isolation of coactivators associated with the TATA-binding protein that mediate transcriptional activation.

A key step in the regulation of transcription involves interactions between promoter-selective factors and various components of the transcriptional apparatus. Here we report the requirements for transcriptional activation directed by NTF-1, a developmentally regulated transcription factor in Drosophila. Reconstituted transcription with fractionated Drosophila basal factors reveals that activation by NTF-1 requires factors present in the endogenous TFIID fraction that are distinct from the purified TATA-binding protein (TBP). Glycerol gradient sedimentation and immunoprecipitation analyses indicate that TFIID is a multiprotein complex containing TBP and at least six tightly bound TBP-associated factors (TAFs). Preparations of TBP lacking TAFs after fractionation with denaturants no longer support activation by NTF-1 but retain basal level activity. Addition of immunopurified and renatured TAFs to free TBP restores the ability of NTF-1 to activate transcription without influencing basal transcription. These results suggest that one or more of the TAF polypeptides confer coactivator function.

Animals↗
PubMed1991

v-Src and EJ Ras alleviate repression of c-Jun by a cell-specific inhibitor.

The AP-1 family of transcription factors, which includes the proto-oncogene products c-Jun and c-Fos, controls the stimulation of cellular genes by growth factors and the expression of oncogenes, including src and ras. Transcriptional activation by c-Jun is regulated by a cell-type-specific inhibitor that represses the activity of a transcriptional activation domain (A1) of c-Jun by operating through the adjacent negative regulatory region (delta). Here we show that cotransfection of the src or ras oncogene enhances the transcriptional activity of a GAL4:c-Jun hybrid that includes the delta-A1 region of c-Jun, suggesting that the DNA binding and dimerization domain of c-Jun is not required for stimulation by Src or Ras. Moreover, induction of c-Jun activity by Src and Ras occurs in cell lines containing the c-Jun inhibitor but not in a cell line lacking it. The region in c-Jun essential for the stimulatory action of these oncogenes maps to domain A1. These findings suggest the existence of signal-transduction pathways that result in an increase in transcriptional activity of c-Jun and AP-1 by disrupting the c-Jun:inhibitor interaction.

Animals↗
PubMed1991

Direct interaction between Sp1 and the BPV enhancer E2 protein mediates synergistic activation of transcription.

The physical interaction of heterologous site-specific DNA-binding proteins is an important theme in eukaryotic transcriptional regulation. In this paper, we show that the cellular transcription factor Sp1 and the BPV-1 (bovine papillomavirus type 1) enhancer protein E2 activate transcription synergistically from two papilloma viral promoters and a series of synthetic promoter constructs in transient transfection experiments. Furthermore, Sp1 can target E2 to a promoter region even in the absence of a specific E2 DNA-binding motif. Biochemical experiments establish that Sp1 enhances E2 binding to its sites and that the two proteins form a specific complex. Sp1 sequesters distally bound E2 to the promoter region by formation of stable DNA loops, visualized by electron microscopy. These experiments substantiate the notion that enhancer binding proteins are targeted to promoter regions by direct interaction with proteins that bind proximal to the transcriptional start site.

Animals↗