Reconstitution of TATA-binding protein-associated factor/TATA-binding protein complexes for in vitro transcription.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R Tjian
Publications and source records attributed to R Tjian.
A nuclear traffic jam: unraveling multicomponent machines and compartments.
Explore the source record for details and available documents.
Animals↗
TAFs mediate transcriptional activation and promoter selectivity.
The TATA-binding protein (TBP)-associated factors (TAFs) of TFIID play a central role in RNA polymerase II transcriptional regulation. Some TAFs can function as co-activators that mediate the activation signal from enhancer-bound regulators. In addition, interactions between selected TAFs and core elements direct promoter selectivity by RNA polymerase II.
Animals↗
Multiple TAFIIs directing synergistic activation of transcription.
Coordinate activation of transcription by multiple enhancer binding factors is essential for the regulation of pattern formation during development of Drosophila melanogaster. Cell-free transcription reactions are described that recapitulate transcriptional synergism directed by the Drosophila developmental regulators Bicoid (BCD) and Hunchback (HB). Within the basal transcription factor complex TFIID, two specific targets, TAFII110 and TAFII60, served as coactivators to mediate transcriptional activation by these two enhancer binding proteins. A quadruple complex containing TATA binding protein (TBP), TAFII250, TAFII110, and TAFII60 mediated transcriptional synergism by BCD and HB, whereas triple TBP-TAFII complexes lacking one or the other target coactivator failed to support synergistic activation. Deoxyribonuclease I footprint protection experiments revealed that an integral step leading to transcriptional synergism involves the recruitment of TBP-TAFII complexes to the promoter by way of multivalent contacts between activators and selected TAFIIs. Thus, the concerted action of multiple regulators with different coactivators helps to establish the pattern and level of segmentation gene transcription during Drosophila development.
Animals↗
DNA template and activator-coactivator requirements for transcriptional synergism by Drosophila bicoid.
The template and coactivator requirements for synergistic transcription directed by a single activator, Bicoid (BCD), bound to multiple sites have been determined. Mutagenesis studies in combination with protein binding experiments and reconstituted transcription reactions identified two independent activation domains of BCD that target different coactivator subunits (TAFII110 and TAFII60) of the basal transcription factor IID (TFIID). The presence of both coactivators is required for BCD to recruit the TATA binding protein (TBP)-TAF complex to the promoter and direct synergistic activation of transcription. Thus, contact between multiple activation domains of BCD and different targets within the TFIID complex can mediate transcriptional synergism.
Animals↗
Coactivator and promoter-selective properties of RNA polymerase I TAFs.
Human ribosomal RNA synthesis by RNA polymerase I requires the activator UBF and the promoter selectivity factor SL1, which consists of the TATA binding protein (TBP) and three associated subunits, TAFI110, TAFI63, and TAFI48. Here it is shown that both TAFI110 and TAFI63 contact the promoter, whereas TAFI48 serves as a target for interaction with UBF and is required to form a transcriptionally active SL1 complex responsive to UBF in vitro. TAFI48 also alters the ability of TBP to interact with TATA box elements, and the resulting complex fails to support transcription by RNA polymerase II. Thus, TAFI48 may function both as a target to mediate UBF activation and as a class-specific promoter selectivity factor.
DNA-Binding Proteins↗
Human TAFII250 interacts with RAP74: implications for RNA polymerase II initiation.
Accurate and regulated transcription by RNA polymerase II requires the assembly of an initiation complex involving multiple protein-DNA and protein-protein interactions. A key event is binding of TFIID, a complex consisting of TBP and associated factors (TAFs) to the template DNA. The TAF subunits of TFIID carry out diverse functions critical for transcription, including specific contact with enhancer proteins and binding to core promoter DNA. However, the role of TAFs in RNA polymerase II-mediated transcription initiation and cross talk with other basal factors remains poorly characterized. Here, we report the specific interaction of TAFII250 with RAP74, an essential subunit of the basal transcription factor IIF. Using various in vitro binding assays we have mapped recognition interfaces between TAFII250 and RAP74. In vivo complementation of a temperature-sensitive TAFII250 cell line reveals that the RAP74 interaction is critical for cell viability. Because TFIIF is thought to be responsible for binding and recruiting RNA polymerase II, the ability of TAFII250 to interact selectively with RAP74 is likely to contribute a critical contact for the assembly of an active transcription complex.
Amino Acids↗
TAFs and TFIIA mediate differential utilization of the tandem Adh promoters.
The D. melanogaster alcohol dehydrogenase (Adh) gene is transcribed from two tandem promoters that are differentially utilized at various stages during development. To determine the mechanism of promoter selectivity, we have analyzed the activity of the Adh promoters both in vitro and in transfected cells. We found that selective promoter utilization is controlled by distinct initiator elements. Reconstitution of Adh transcription with purified components requires a specific TBP-TAF complex that, in concert with TFIIA, directs differential Adh promoter transcription. Fractionation of this TBP-TAF complex reveals that TAFII150 is required for discrimination between the proximal and distal promoters. We propose a mechanism for regulating differential promoter utilization during Drosophila development that involves the recognition of specific initiator elements by TAFs in the TFIID complex.
Alcohol Dehydrogenase↗
Synergistic activation of transcription by UNC-86 and MEC-3 in Caenorhabditis elegans embryo extracts.
The nematode Caenorhabditis elegans has been a choice organism for the study of developmental regulation using classical and molecular genetic approaches. Consequently, many genetically defined pathways have been described and numerous regulatory genes have been identified. However, the biochemical and functional properties of these putative transcription factors have remained uncharacterized, partly because C.elegans cell-free transcription reactions have not been developed. Here we describe the in vitro transcriptional activation properties of two C.elegans homeodomain proteins, UNC-86 and MEC-3, in nuclear extracts derived from C.elegans embryos. Whereas the POU homeodomain protein, UNC-86, alone was able to activate transcription of the mec-3 promoter in vitro, the LIM homeodomain protein, MEC-3, failed to bind DNA or activate transcription on its own. However, in the presence of both UNC-86 and MEC-3, we observed cooperative binding to the mec-3 promoter and synergistic activation of transcription in vitro. Protein-protein interaction assays revealed that UNC-86 can bind directly to MEC-3, and in vitro transcription studies indicate that both proteins contain a functional activation domain. Thus, formation of a heteromeric complex containing two activation domains results in a highly potent activator. These studies provide direct functional evidence for coordinated transcriptional activation by two C.elegans DNA binding proteins that have been defined genetically as regulators of gene expression during embryogenesis.
Animals↗
Functional domain analysis of glass, a zinc-finger-containing transcription factor in Drosophila.
The glass gene is required for proper photo-receptor differentiation during development of the Drosophila eye glass codes for a DNA-binding protein containing five zinc fingers that we show is a transcriptional activator. A comparison of the sequences of the glass genes from two species of Drosophila and a detailed functional domain analysis of the Drosophila melanogaster glass gene reveal that both the DNA-binding domain and the transcriptional-activation domain are highly conserved between the two species. Analysis of the DNA-binding domain of glass indicates that the three carboxyl-terminal zinc fingers alone are necessary and sufficient for DNA binding. We also show that a deletion mutant of glass containing only the DNA-binding domain can behave in a dominant-negative manner both in vivo and in a cell culture assay that measures transcriptional activation.
Amino Acid Sequence↗
Binding of TAFs to core elements directs promoter selectivity by RNA polymerase II.
The mechanisms that govern core promoter recognition and basal transcription efficiency remain poorly understood. Here, we have assessed the potential role of TAFs and the TFIID complex in directing basal promoter function. Reconstituted transcription reactions revealed the ability of TFIID versus TBP to discriminate between distinct core promoters. A comparison of different partial TBP-TAF assemblages established that a trimeric TBP-TAFII250-TAFII150 complex is minimally required for efficient utilization of the initiator and downstream promoter elements. Depending on the promoter structure, TAFs can increase or decrease the stability of TFIID-promoter interactions. These findings suggest that TAFs play a critical role in promoter selectivity and transcription regulation through direct contacts with core promoter elements.
Amino Acid Sequence↗
Molecular cloning and expression of the 32-kDa subunit of human TFIID reveals interactions with VP16 and TFIIB that mediate transcriptional activation.
Transcription factor TFIID consists of TATA binding protein (TBP) and at least eight TBP-associated factors (TAFs). As TAFs are required for activated but not basal transcription, we have proposed that TAFs act as coactivators to mediate signals between activators and the basal transcription machinery. Here we report the cloning, expression, and biochemical characterization of the 32-kDa subunit of human (h) TFIID, termed hTAFII32. We find that hTAFII32 is the human homologue of Drosophila TAFII40. In vitro protein-protein interaction assays reveal that as observed with Drosophila TAFII40, hTAFII32 interacts with the C-terminal 39-amino acid activation domain of the acidic transactivator viral protein 16 (VP16) as well as with the general transcription factor TFIIB. Moreover, a partial recombinant TFIID complex containing hTAFII32 was capable of mediating in vitro transcriptional activation by the VP16 activation domain. These findings indicate that specific activator-coactivator interactions have been conserved between human and Drosophila and provide additional support for the function of these interactions in mediating transcriptional activation.
Amino Acid Sequence↗
p53 transcriptional activation mediated by coactivators TAFII40 and TAFII60.
The tumor suppressor protein p53 is a transcriptional regulator that enhances the expression of proteins that control cellular proliferation. The multisubunit transcription factor IID (TFIID) is thought to be a primary target for site-specific activators of transcription. Here, a direct interaction between the activation domain of p53 and two subunits of the TFIID complex, TAFII40 and TAFII60, is reported. A double point mutation in the activation domain of p53 impaired the ability of this domain to activate transcription and, simultaneously, its ability to interact with both TAFII40 and TAFII60. Furthermore, a partial TFIID complex containing Drosophila TATA binding protein (dTBP), human TAFII250, dTAFII60, and dTAFII40 supported activation by a Gal4-p53 fusion protein in vitro, whereas TBP or a subcomplex lacking TAFII40 and TAFII60 did not. Together, these results suggest that TAFII40 and TAFII60 are important targets for transmitting activation signals between p53 and the initiation complex.
Amino Acid Sequence↗
Molecular machines that control genes.
Explore the source record for details and available documents.
Animals↗
Reconstitution of transcription factor SL1: exclusive binding of TBP by SL1 or TFIID subunits.
RNA polymerase I and II transcription factors SL1 and TFIID, respectively, are composed of the TATA-binding protein (TBP) and a set of TBP-associated factors (TAFs) responsible for promoter recognition. How the universal transcription factor TBP becomes committed to a TFIID or SL1 complex has not been known. Complementary DNAs encoding each of the three TAFIs that are integral components of SL1 have not been isolated. Analysis of subunit interactions indicated that the three TAFIs can bind individually and specifically to TBP. In addition, these TAFIs interact with each other to form a stable TBP-TAF complex. When TBP was bound first by either TAFI110, 63, or 48, subunits of TFIID such as TAFII250 and 150 did not bind TBP. Conversely, if TBP first formed a complex with TAFII250 or 150, the subunits of SL1 did not bind TBP. These results suggest that a mutually exclusive binding specificity for TBP intrinsic to SL1 and TFIID subunits directs the formation of promoter- and RNA polymerase-selective TBP-TAF complexes.
Amino Acid Sequence↗
Assembly of transcriptionally active RNA polymerase I initiation factor SL1 from recombinant subunits.
Initiation of ribosomal RNA synthesis by RNA polymerase I requires the promoter selectivity factor SL1, which consists of the TATA-binding protein, TBP, and three associated factors, TAFIS 110, 63, and 48. Here the in vivo and in vitro assembly of functional SL1 complexes from recombinant TAFIS and TBP are reported. Complexes containing TBP and all three TAFIS were as active in supporting transcription from the human ribosomal RNA gene promoter as endogenous SL1, whereas partial complexes without TBP did not efficiently direct transcription in vitro. These results suggest that TAFIS 110, 63, and 48, together with TBP, are necessary and sufficient to reconstitute a transcriptionally active SL1 complex.
DNA-Binding Proteins↗
Multiple regions of TBP participate in the response to transcriptional activators in vivo.
We used mutant yeast and human TBP molecules with an altered DNA-binding specificity to examine the role of TBP in transcriptional activation in vivo. We show that yeast TBP is functionally equivalent to human TBP for response to numerous transcriptional activators in human cells, including those that do not function in yeast. Despite the extensive conservation of TBP, its ability to respond to transcriptional activators in vivo is curiously resistant to clustered sets of alanine substitution mutations in different regions of the protein, including those that disrupt DNA binding and basal transcription in vitro. Combined sets of these mutations, however, can attenuate the in vivo activity of TBP and can differentially affect response to different activation domains. Although the activity of TBP mutants in vivo did not correlate with DNA binding or basal transcription in vitro, it did correlate with binding in vitro to the largest subunit of TFIID, hTAFII250. Together, these data suggest that TBP utilizes multiple interactions across its surface to respond to RNA polymerase II transcriptional activators in vivo; some of these interactions appear to involve recruitment of TBP into TFIID, whereas others are involved in response to specific types of transcriptional activators.
Gene Expression Regulation↗
Assembly of recombinant TFIID reveals differential coactivator requirements for distinct transcriptional activators.
We previously reported that transcriptional regulators can bind selected TAF subunits of the TFIID complex. However, the specificity and function of individual TAFs in mediating transcriptional activation remained unknown. Here we report the in vitro assembly and transcriptional properties of TBP-TAF complexes reconstituted from the nine recombinant subunits of Drosophila TFIID. A minimal complex containing TBP and TAFII250 directs basal but not activator-responsive transcription. By contrast, reconstituted holo-TFIID supports activation by an assortment of activators. The activator NTF-1, which binds TAFII150, stimulates transcription with a complex containing only TBP, TAFII250, and TAFII150, whereas Sp1 binds and additionally requires TAFII110 for activation. Interestingly, TAFII150 enhances Sp1 activation even though this subunit does not bind directly to Sp1. These results establish that specific subcomplexes of TFIID can mediate activation by different classes of activators and suggest that TAFs perform multiple functions during activation.
Amino Acid Sequence↗