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A J Berk

Publications and source records attributed to A J Berk.

At least 55 records · Page 3Linked to original sources
PubMed1993

The p53 activation domain binds the TATA box-binding polypeptide in Holo-TFIID, and a neighboring p53 domain inhibits transcription.

Antioncogene product p53 is a transcriptional transactivator. To investigate how p53 stimulates transcription, we examined the interaction of p53 with general transcription factors in vitro. We found that p53 binds directly to the human TATA box-binding polypeptide (TBP). We also observed a direct interaction between p53 and purified holo-TFIID, a complex composed of TBP and a group of TBP-associated polypeptides known as TAFs. The p53 binding domain on TBP was mapped to the conserved region of TBP, including residues 220 to 271. The TBP binding domain on p53 was mapped to the p53 activation domain between residues 20 and 57. To analyze the significance of the p53-TBP interaction in p53 transactivation, we compared the ability of Gal4-p53 fusion proteins to bind to TBP in vitro and to activate transcription in transient transfection assays. Fusion proteins which bound to TBP activated transcription, and those that did not bind to TBP did not activate transcription to a detectable level, suggesting that a direct interaction between TBP and p53 is required for p53 transactivation. We also found that inclusion of residues 93 to 160 of p53 in a Gal4-p53 fusion repressed transcriptional activation 100-fold. Consequently, this region of p53 inhibits transcriptional activation by the minimal p53 activation domain. Highest levels of activation were observed with sequences 1 to 92 of p53 fused to Gal4, even though this construct bound to TBP in vitro with an affinity similar to that of other Gal4-p53 fusion proteins. We conclude that TBP binding is necessary for p53 transcriptional activation and that p53 sequences outside the TBP binding domain modulate the level of activation.

Amino Acid Sequence↗
PubMed1992

Inhibition of p53 transactivation required for transformation by adenovirus early 1B protein.

The cellular phosphoprotein p53 inhibits progression through the mammalian cell cycle. Both p53 alleles are frequently mutated in human tumours, indicating that p53 is a tumour suppressor. Recent studies have suggested that p53 functions as a transcriptional activator, but the significance of this activity in cell-cycle control has not been established. The adenovirus 2 (Ad2) early 1B (E1B) 55K protein binds to p53 in transformed cells and contributes to oncogenic transformation by Ad2 (refs 10-12). Here we report that mutants of E1B 55K and wild-type Ad12 E1B 54K proteins show a strong correlation between their ability to inhibit p53-mediated transcriptional activation and their ability to cooperate with adenovirus E1A protein in the transformation of primary cells. These results indicate that p53 probably inhibits cell cycling by functioning as a transcription factor.

Adenovirus Early Proteins↗
PubMed1992

Holo-TFIID supports transcriptional stimulation by diverse activators and from a TATA-less promoter.

Transcription factor IID (TFIID) binds to TATA boxes, nucleating the assembly of initiation complexes containing several general transcription factors and RNA polymerase II. Recently, TFIID was shown to be a multisubunit complex containing a TATA box-binding polypeptide (TBP) and several tightly associated polypeptides (TAFs), which are required for transcriptional stimulation by activator proteins. Here, we report the development of a human cell line expressing an epitope-tagged TBP and the immunopurification of a native, high-molecular-weight form of TFIID that supports transcriptional stimulation by several different classes of activation domains. Recovery of basal and activated TFIID transcriptional specific activity was close to approximately 100%. Electrophoretic mobility-shift analysis demonstrated a single major DNA-protein complex. This holo-TFIID contains TAFs of approximately 250, 125, 95, 78, and 50 kD and sediments at 17S. Holo-TFIID produced an extended footprint over the adenovirus major late promoter TATA box and initiator sequence and supported transcriptional activation from a promoter lacking a TATA box. These results lead us to hypothesize that a single multisubunit TFIID protein supports transcriptional stimulation by diverse activation domains and from a TATA-less promoter.

Base Sequence↗
PubMed1991

Promoter activity and distance constraints of one versus two Sp1 binding sites.

In the simple adenovirus 2 early 1B (E1B) promoter, moving the single Sp1 transcription factor binding site (GC box) from its wild-type position eight base pairs (bp) from the TATA box to 30 bp upstream is equivalent to its deletion (Wu, L., and Berk, A. (1988) Genes & Dev. 2, 403-411). In more complex promoters, multiple upstream elements regulate transcription over greater distances. To understand these spacing constraints, we placed two GC boxes in phase at various distances from the E1B TATA box. Whereas one GC box at eight bp from TATA increases transcription in vivo 5-fold compared with TATA alone (Wu, L., Rosser, D. S. E., Schmidt, M. C., and Berk, A. (1987) Nature 326, 512-515), two GC boxes increased in vivo transcription 25-30-fold. Transcriptional stimulation by two GC boxes fell off rapidly in vivo at 30 bp from TATA, remained approximately constant through 50 bp, then decreased again at 70 bp. Consequently, two GC boxes have a multiplicative effect at eight bp from TATA and continue to stimulate transcription at a greater distance from TATA than a single Sp1 site. Quantitatively different results were observed for in vitro transcription using a nuclear extract; a more linear fall off with increasing distance from TATA was observed. Separating the two GC boxes progressively decreased transcription. E1A stimulation of these promoters in vivo indicates that Sp1 transcription control is independent of E1A transactivation.

Adenoviridae↗
PubMed1991

Adenovirus E1A activation domain binds the basic repeat in the TATA box transcription factor.

The adenovirus large E1A protein is a potent activator of transcription. We use several different experimental approaches to demonstrate that the large E1A protein binds specifically and stably to the TATA box-binding factor (TFIID), the general polymerase II transcription factor that initiates assembly of transcription complexes. Sedimentation velocity centrifugation revealed that TFIID and E1A form a heterodimer in vitro. We demonstrate that the activation domain of E1A (conserved region 3) binds to TFIID. E1A interacts with a 51 residue region from the conserved C-terminal domain of TFIID that includes a repeat of basic residues between the homologous direct repeats of TFIID. Analysis of TFIID binding by various E1A mutants indicates that TFIID binding is necessary, although not sufficient, for E1A transactivation.

Adenoviridae↗
PubMed1991

Requirement for acidic amino acid residues immediately N-terminal to the conserved domain of Saccharomyces cerevisiae TFIID.

TFIID binds to TATA boxes and initiates the assembly of general transcription factors and pol II on promoters. TFIID proteins from various species consist of a highly conserved carboxy terminal domain and very divergent amino terminal domains. We investigated the function of the non-conserved amino terminal domain (residues 1-60) of Saccharomyces cerevisiae TFIID (YIID, 240 residues) by testing the ability of a series of YIID amino terminal deletion mutants to complement a YIID deficient yeast strain. Mutants with deletions up to amino acid 48 restored the YIID deficient yeast strain to an apparently wild type phenotype. However, deletion up to position 57 or 60 produced yeast strains which formed extremely small colonies. Moreover, overexpression of YIID delta 2-57 or YIID delta 3-60 protein in the presence of wild type YIID resulted in a dominant-negative inhibition of growth. No difference between the basal transcriptional activity of wild type YIID and these amino terminal deletion mutants was observed in vitro. However, transcriptional activation in vivo of promoter-lacZ fusions showed that the YIID delta 2-57 deletion affects the ability of certain promoters (CUP1 and an HSP UAS-CYC1 promoter hybrid promoter) to respond to upstream factor stimulation. At least one inducible promoter, PHO5, was not affected by this deletion. The defect produced by YIID delta 2-57 was due to the deletion of several acidic residues present between residues 48 and 57. The results show that the conserved carboxy terminal domain of YIID is sufficient for cell viability. However, an acidic region just amino terminal to the conserved domain is required for normal growth and transcription control in most yeast strains.

Amino Acid Sequence↗
PubMed1991

Affinity purification of transcription factor IIA from HeLa cell nuclear extracts.

One of the general transcription factors, TFIIA, was purified to homogeneity from HeLa cell nuclear extracts by yeast TFIID affinity chromatography. Human TFIIA had a molecular weight of approximately 38 kd. It was able to associate with the complex formed by yeast TFIID and the TATA elements of the adenovirus E4 and ML promoters, and the HSP70 promoter. The association extended the protected region on each TATA element by yeast TFIID from DNase I digestion. Affinity-purified TFIIA was also able to stimulate transcription from the E4 and ML promoters in in vitro reconstituted systems.

Adenoviridae↗
PubMed1991

The Zta trans-activator protein stabilizes TFIID association with promoter DNA by direct protein-protein interaction.

Eukaryotic transcriptional activators are believed to stimulate transcription through direct and/or indirect interactions with one or more of the general transcription factors. We show here that the Zta transcriptional activator protein encoded by the Epstein-Barr virus makes direct physical contact with the basic transcription factor TFIID. Both Zta and TFIID were expressed in and purified from Escherichia coli. Zta stabilized the binding of TFIID to Zta-responsive promoters as assayed by gel electrophoresis mobility-shift and immunoprecipitation of radiolabeled promoter DNA. A deletion mutant of Zta that failed to activate transcription failed to stabilize TFIID binding. DNase I footprinting showed that Zta reduced the dissociation rate of TFIID bound to the TATA element. Protein blotting and immunoprecipitation experiments demonstrated that TFIID and Zta also interact in the absence of promoter DNA. The amino acid residues 25-86 of Zta were essential for the stable association with TFIID and were shown to be required for trans-activation in vivo. We propose that Zta stimulates transcription, in part, by direct physical contact with the conserved domain of TFIID and the formation of a stable Zta-TFIID-promoter complex.

Amino Acid Sequence↗
PubMed1991

Two distinct domains in the yeast transcription factor IID and evidence for a TATA box-induced conformational change.

Transcription factor IID from Saccharomyces cerevisiae (YIID) binds the TATA box element present in most RNA polymerase II promoters. In this work, partial proteolysis was used as a biochemical probe of YIID structure. YIID consists of a protease-sensitive amino terminus and a highly stable, protease-resistant carboxy-terminal core. The cleavage sites of the predominant chymotrypsin- and trypsin-derived fragments were mapped to amino acid residues 40 to 41 and 48 to 49, respectively, by amino-terminal peptide sequencing. Removal of the amino terminus resulted in a dramatic increase in the ability of YIID to form a stable complex with DNA during gel electrophoresis mobility shift assays and a two- to fourfold increase in DNA-binding affinity, as assayed by DNase I footprinting analysis. The carboxy-terminal 190-amino-acid core was competent for transcription in vitro and was similar in activity to native YIID. DNA containing a TATA element induced hypersensitive sites in the amino-terminal domain and stabilized the core domain to further proteolytic attack. Native YIID did not bind to a TATA box at 0 degrees C, whereas the carboxy-terminal DNA-binding domain did. These results suggest that YIID undergoes a conformational change upon binding to a TATA box. Southern blotting showed that the carboxy-terminal domain is highly conserved, while the amino-terminal domain diverged rapidly in evolution, even between closely related budding yeasts.

Binding Sites↗
PubMed1990

Cloning of a transcriptionally active human TATA binding factor.

Transcription factor IID (TFIID) binds to the TATA box promoter element and regulates the expression of most eukaryotic genes transcribed by RNA polymerase II. Complementary DNA (cDNA) encoding a human TFIID protein has been cloned. The human TFIID polypeptide has 339 amino acids and a molecular size of 37,745 daltons. The carboxyl-terminal 181 amino acids of the human TFIID protein shares 80% identity with the TFIID protein from Saccharomyces cerevisiae. The amino terminus contains an unusual repeat of 38 consecutive glutamine residues and an X-Thr-Pro repeat. Expression of DNA in reticulocyte lysates or in Escherichia coli yielded a protein that was competent for both DNA binding and transcription activation.

Amino Acid Sequence↗
PubMed1990

Dissection of functional domains in the adenovirus 2 early 1B 55K polypeptide by suppressor-linker insertional mutagenesis.

To determine whether the viral replication functions of the adenovirus E1B 55K protein play a role in its ability to transform cloned rat embryo fibroblast cells in culture, we constructed an extensive series of insertion mutations throughout the 55K gene. The mutations were recombined into infectious virus and characterized for their abilities to produce stable 55K protein in HeLa cells, replicate virus in HeLa cells, express late viral proteins efficiently, and transform CREF cells following infection. Mutant 55K transforming activity in primary baby rat kidney cells was also assayed following DNA transfection. The functions required for viral replication are encoded in several patches of the 55K linear sequence, while the CREF transforming functions are sensitive to all of the insertions. An insertion at amino acid 380 created a mutant virus which was reduced in transforming activity, but was not reduced for viral replication. Therefore, a function required for efficient transformation of CREF cells can be separated from functions required for late gene expression and viral replication. Transformation of BRK cells following DNA transfection was reduced by complete disruption of the 55K protein gene, but was not significantly affected by any of the insertions.

Adenovirus Early Proteins↗
PubMed1990

Domains required for in vitro association between the cellular p53 and the adenovirus 2 E1B 55K proteins.

The 55K protein encoded by the adenovirus 2 E1B gene is required for complete cellular transformation and binds the cellular protein p53. Using an in vitro immunoprecipitation assay, we mapped the domains in both 55K and p53 required for the interaction of the two proteins. The domain in p53 mapped to the amino terminal 123 residues. There are several domains in the 495 residue 55K polypeptide which contribute to stable association with p53, with the most essential region mapping between residues 224 and 354. Mutations which prevented 55K-p53 binding were not more defective for transformation than other mutations which did not affect binding.

Adenovirus Early Proteins↗
PubMed1990

Transcriptional activation by Sp1 as directed through TATA or initiator: specific requirement for mammalian transcription factor IID.

Transcription of mammalian genes by RNA polymerase II often begins at a specific nucleotide, whose location is determined either by an upstream DNA element known as a TATA box or by an element positioned at the transcription start site called an initiator (Inr). By in vitro analysis of synthetic promoters, we demonstrate here that the TATA and Inr elements are functionally similar and that the Inr is contained between nucleotides -3 and +5 relative to the initiation site. Moreover, we found that a mammalian transcription factor IID (TFIID) protein fraction is required for transcriptional stimulation by an Sp1-dependent activating element placed upstream of either TATA or Inr elements. However, in these assays, the yeast TATA-binding protein, which previously was shown to function similarly to mammalian TFIID, could not efficiently substitute for the mammalian TFIID fraction. These results demonstrate that mammalian TFIID is functionally distinct from the yeast TATA-binding protein and may contain additional subunits or domains that are important for transcriptional activation from some promoters.

Adenovirus Early Proteins↗
PubMed1990

High-level transcription from the adenovirus major late promoter requires downstream binding sites for late-phase-specific factors.

The adenovirus major late promoter (MLP) is active during both the early and late phases of infection. During the early phase the activity of the MLP is similar to those of the other early viral promoters, but during the late phase the rate of transcription from the MLP becomes much greater by comparison. We report here that sequence-specific binding proteins are induced during the late phase which interact with three regions in the first intron of the MLP transcription unit from positions +37 to +68, +80 to +105, and +105 to +125 relative to the transcription initiation site. To measure the significance of these binding sites for transcription during the late phase, we constructed MLP-beta-globin fusions and substituted them for early region 3 in adenovirus recombinants. Deletion of the binding sites caused significant reductions in the rate of transcription, specifically during the late phase of infection. Deletion of all three sites reduced the rate of transcription 25- to 50-fold and the accumulation of cytoplasmic MLP-beta-globin RNA 200-fold. These results indicate that the high rate of transcription from the MLP during the late phase of infection results from the interaction of virus-induced transcription factors with three binding sites in the first intron of the major late transcription unit.

Adenoviruses, Human↗
PubMed1990

In vitro transcriptional activation, dimerization, and DNA-binding specificity of the Epstein-Barr virus Zta protein.

The Epstein-Barr virus BZLF1 immediate-early gene encodes a transcriptional activator protein, Zta, which acts as a key regulatory switch in the transition between the latent and lytic viral life cycle. In this work, full-length Zta was expressed at high levels in Escherichia coli and purified to homogeneity by DNA affinity chromatography. The bacterial protein bound to specific target sequences (Zta response elements) and activated transcription in vitro from an Epstein-Barr virus early target promoter (BHLF1). Zta bound to DNA as a dimer. The formation of a heterodimer with a Zta deletion mutant was detected by gel electrophoresis mobility shift assays. Footprinting analysis on the BHLF1, BZLF1, and simian virus 40 control regions revealed multiple binding sites with no simple consensus sequence. Zta bound upstream from its own promoter at low concentrations, while at high concentrations it bound at the transcription start site, suggesting that it may activate and then autoregulate its own expression. These results demonstrate that Zta is a sequence-specific DNA-binding transcription factor.

Amino Acid Sequence↗
PubMed1989

A DNA-binding domain of human transcription factor IIIC2.

Transcription factor IIIC2 is required for in vitro transcription of the adenovirus 2 VA1 gene and binds with high affinity to its B-box promoter element which is an 18 bp perfect inverted repeat. Partial proteolysis of TFIIIC2 with chymotrypsin and Staphylococcus aureus V8 protease yielded a species which produced a discrete band in a gel shift assay with about twice the mobility of the undigested complex. Chymotrypsin-digested TFIIIC2 produced a DNase I footprint virtually identical to that of the undigested protein, but the stability of the protein-VA1 DNA complex was drastically reduced and the in vitro transcriptional activity was eliminated. These results indicate that a chymotrypsin-resistant domain of TFIIIC2 binds to the B-box sequence. We speculate that stable binding requires protease sensitive cooperative interactions between TFIIIC2 DNA-binding domains.

Adenoviruses, Human↗
PubMed1989

Purification and characterization of transcription factor IIIC2.

Transcription factor IIIC2 (TFIIIC2), together with other transcription factors (TFIIIB and TFIIIC1), is required for the in vitro transcription of tRNA and adenovirus VA genes by RNA polymerase III. Previous studies have shown that TFIIIC2 is a high molecular weight (approximately 500,000) protein which binds with high affinity to the B-box promoter element of tRNA-type genes. A polypeptide of Mr approximately 250,000 is in close association with DNA in the specific complex between TFIIIC2 and the B-box promoter element. Here we describe the purification of TFIIIC2 by a factor of approximately 25,000 from nuclear extracts of HeLa cells by ionic exchange, affinity, and hydrophobic chromatography and sedimentation velocity centrifugation. The most purified fractions contain polypeptides of approximately 230 kDa (corresponding to the polypeptide which can be cross-linked to VA1 DNA), 110, 100, 80, and 60 kDa which co-sediment with TFIIIC2 B-box specific binding and in vitro transcriptional activities.

Adenoviruses, Human↗