Characterization of RNA molecules by S1 nuclease analysis.
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DNA Probes↗
Biomedical subjects
A J Berk
Publications and source records attributed to A J Berk.
Yeast TATA-box transcription factor gene.
The first step in the transcription of most protein-encoding genes in eukaryotes is the binding of a transcription factor to the TATA-box promoter element. This TATA-box transcription factor was purified from extracts of the yeast Saccharomyces cerevisiae by using reconstitution of in vitro transcription reactions as an assay. The activity copurified with a protein whose sodium dodecyl sulfate/polyacrylamide gel mobility is 25 kDa. The sequence of the amino-terminal 21 residues of this protein was determined by sequential Edman degradation. A yeast genomic library was screened with mixed oligonucleotides encoding six residues of the protein sequence. The yeast TATA-box factor gene was cloned, and DNA sequencing revealed a 720-base-pair open reading frame encoding a 27,016-Da protein. The identity of the clone was confirmed by expressing the gene in Escherichia coli and detecting TATA-box factor DNA binding and transcriptional activities in extracts of the recombinant E. coli. The TATA-box factor gene was mapped to chromosome five of S. cerevisiae. RNA blot hybridization and nuclease S1 analysis indicated that the major TATA-box factor mRNA is 1.3 kilobases, including an unusually long 5' untranslated region of 188 +/- 5 nucleotides. Homology searches showed a region of distant similarity to the calcium-binding structures of calpains, a structure that has a conformation similar to the helix-turn-helix motif of DNA binding proteins.
Amino Acid Sequence↗
Multiple transcription factor binding sites mediate adenovirus E1A transactivation.
We studied the response of simple synthetic promoter regions to transactivation by the adenovirus early region 1A (E1A) protein. Binding sites for one or two host cell transcription factors were substituted for the E1B promoter region in reconstructed virus mutants, and the response to E1A transactivation was assayed during the early phase of infection. We found that a single CREB/ATF binding site resulted in a surprisingly strong promoter which responded to E1A. A CREB/ATF binding site placed upstream of the E1B TATA box behaved much like the wild-type E1B promoter, which is composed of a single Sp1 binding site plus a TATA box. A single E2F binding site resulted in an extremely weak promoter which did not respond to E1A, much like a single Sp1 site. Two E2F sites in an inverted orientation with the same spacing as in the adenovirus type 2 E2 early promoter produced a strong, E1A-responsive promoter. Substitution of the E4 TATA box region for the E1B TATA box region produced a promoter about five times stronger than the wild-type E1B promoter in the absence of E1A. However, the E4 TATA box substitution did not respond significantly to E1A transactivation. These results directly demonstrate that many different transcription factor binding sites, including the E1B TATA box, a CREB/ATF binding site, and two E2F sites, can mediate E1A transactivation. Other transcription factor binding sites cannot mediate an E1A response; these other sites include the E4 TATA box, a single Sp1 binding site, and a single E2F binding site. Implications of these findings for the mechanism of E1A transactivation are discussed.
Adenovirus Early Proteins↗
Human transcription factor TFIIIC2 specifically interacts with a unique sequence in the Xenopus laevis 5S rRNA gene.
Transcription factor TFIIIC2 derived from human cells is required for tRNA-type gene transcription and binds with high affinity to the essential B-box promoter element of tRNA-type genes. Although 5S rRNA genes contain no homology with the tRNA-type gene B box, we show that TFIIIC2 is also required for Xenopus laevis 5S rRNA gene transcription. TFIIIC2 protected an approximately 30-base-pair (-10 to +18) region of a Xenopus 5S rRNA gene from DNase I digestion. This region, which spanned the transcription start site, included sequences that are highly conserved among eucaryotic 5S rRNA genes and have no homology with the B-box sequence of tRNA genes. Mutation of the TFIIIC2-binding site reduced transcription of the 5S rRNA gene by a factor of 10 in HeLa cell extracts. Methylation of C residues within the TFIIIC2-binding site interfered with binding of TFIIIC2. These results suggest a role of the TFIIIC2-binding sequence in 5S rRNA gene transcription. In addition, the 5S rRNA gene binding site and the tRNA-type gene B-box sequence did not compete with each other for binding to TFIIIC2 any better than did an unrelated DNA sequence, indicating that TFIIIC2 interacts with 5S rRNA genes and tRNA-type genes through separate DNA-binding domains or polypeptides.
Animals↗
Sp1 activates transcription without enhancing DNA-binding activity of the TATA box factor.
We have studied the interactions of the Sp1 and IID transcription factors with a simple RNA polymerase II promoter. The adenovirus E1B core promoter consists essentially of a GC box and a TATA box, binding sites for the Sp1 and IID transcription factors, respectively. The E1B promoter is accurately transcribed in vitro using a mammalian transcription system. Sp1 activates E1B transcription in vitro in reactions using IID factor isolated from either human or yeast cells. In DNase I footprinting studies, Sp1 bound rapidly to its recognition sequence even at 0 degrees C (t1/2 less than 1 min). In contrast, yeast IID bound more slowly (t1/2 approximately 6 min at 25 degrees C) and required thermal energy for stable binding to the TATA box sequence. Dissociation rates were measured by the addition of specific oligonucleotide competitors to preformed DNA-protein complexes. Sp1 dissociates rapidly (t1/2 less than 1 min) at 25 degrees C, while yeast IID dissociates with an estimated t1/2 of 1 h at 25 degrees C. Sp1 and yeast IID bound to the E1B promoter simultaneously but independently. The rates of binding and dissociation of these factors were not significantly affected by the presence of the other factor. Bound Sp1 factor did not alter or enhance the yeast IID footprint. Oligonucleotide challenge of in vitro transcription reactions indicated that Sp1 also did not enhance the binding of the human IID factor to the E1B promoter. Thus the Sp1 factor activates transcription of the E1B gene by a mechanism that does not enhance the DNA-binding activity of the IID factor. Sp1 factor activates E1B transcription by 5- to 10-fold in vitro. Under these in vitro transcription conditions, transcripts due to reinitiation from an individual promoter complex contribute only a small portion of the total yield of E1B transcripts. Thus Sp1 cannot activate transcription by increasing the rate of initiation events per complex. Instead it appears that Sp1 acts by increasing the number of productive transcription complexes formed in vitro.
Adenoviridae↗
Transcriptional activation by the pseudorabies virus immediate early protein requires the TATA box element in the adenovirus 2 E1B promoter.
The adenovirus E1A protein and immediately early (IE) protein of pseudorabies virus stimulate transcription from many promoters. It has been postulated that these proteins function via cellular intermediates. We analyzed the transcription of adenovirus E1B promoter mutants in response to the stimulatory effect of IE protein in comparison with E1A. Far upstream deletions and precise Sp1 and TATA box substitution mutations were examined. Only mutations affecting the TATA box element interfered with transactivation by IE protein. This result is similar to observations for E1A transactivation (L. Wu et al. Nature (London) 326, 512-515, 1987), suggesting that both E1A and IE proteins mediate transactivation of the E1B promoter through a common cellular intermediate which interacts with the TATA box.
Adenoviruses, Human↗
Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.
The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.
Adenovirus Early Proteins↗
Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.
Extracts of adenovirus-infected HeLa cells have 5- to 10-fold-higher activity for transcription from the major late promoter in vitro than do extracts of mock-infected or E1A mutant-infected cells (K. Leong and A. J. Berk, Proc. Natl. Acad. Sci. USA 83:5844-5848, 1986). In this study, we analyzed extracts from mock-infected cells and from cells infected with an E1A mutant, pm975, which expresses principally the large E1A protein responsible for the stimulation of transcription. These extracts were fractionated by phosphocellulose chromatography, a procedure which separates factors required for transcription from this promoter (J. D. Dignam, B. S. Shastry, and R. G. Roeder, Methods Enzymol. 101:582-589, 1983), allowing the quantitative assay of individual factors (M. Samuels, A. Fire, and P. A. Sharp, J. Biol. Chem. 257:14419-14427, 1982). Fractions eluted with 0.04, 0.35, and 0.6 M KCl, which contained RNA polymerase II, the upstream factor MLTF, and three general polymerase II transcription factors, had similar activities when prepared from virus-infected or from mock-infected cells. The sequence-specific DNA-binding activity of MLTF was also similar in the virus-infected- and mock-infected-cell extracts. In contrast, the 1.0 M KCl fraction prepared from virus-infected cells consistently exhibited activity severalfold higher than that of the equivalent fraction prepared in parallel from mock-infected cells. E1A protein eluted principally (greater than 80%) in the 0.35 M KCl fraction. Results of others (M. Sawadogo and R. G. Roeder, Cell 43:165-175, 1985) have shown that the 1.0 M KCl fraction, containing 2 to 5% of the unfractionated protein extract, contains a factor which binds specifically to the major late promoter TATA box. These results, together with a recent genetic analysis of the E1B promoter which demonstrated that the TATA box was required for its efficient transcriptional activation (transactivation) by E1A (L. Wu, D. S. E. Rosser, M. Schmidt, and A. J. Berk, Nature (London) 326:512-515, 1987), are consistent with the model that E1A protein indirectly activates the TATA box transcription factor. Consistent with this model was the finding that mutants of the major late promoter containing only the TATA box and cap site region were transcribed at higher rates with extracts from virus-infected cells than with extracts from mock-infected cells. Other models consistent with the results are also discussed.
Adenovirus Early Proteins↗
Ordering promoter binding of class III transcription factors TFIIIC1 and TFIIIC2.
The separation of the mammalian class III transcription factor TFIIIC into two functional components, termed TFIIIC1 and TFIIIC2, enabled an analysis of their functions in transcription initiation. Template competition assays were used to define the order with which these factors interact in vitro to form stable preinitiation complexes on the adenovirus VAI and Drosophila melanogaster tRNA(Arg) genes. The interaction between these genes and TFIIIC2, the factor that binds with high affinity to the B block, was both necessary and sufficient for template commitment. When either the VAI or tRNA(Arg) gene was preincubated with TFIIIC2 alone, transcription of a second gene added subsequently was excluded, indicating that TFIIIC2 bound stably to the first template. Furthermore, the interaction between TFIIIC2 and these genes must occur prior to that of TFIIIC1 or TFIIIB. Once TFIIIC2 was bound, TFIIIC1 could bind to the tRNA(Arg) and VAI genes, although its interaction with the VAI gene was less stable than that with the tRNA(Arg) gene. TFIIIB activity bound stably to the complex of both genes with TFIIIC2. These results demonstrate that TFIIIC2 is the first transcription factor to bind to these genes and that TFIIIB and TFIIIC1 can then interact in either order to form a preinitiation complex.
Adenoviridae↗
Separation of TFIIIC into two functional components by sequence specific DNA affinity chromatography.
Recently, it has been shown that mammalian transcription factor IIIC (TFIIIC) activity can be separated by anion exchange FPLC chromatography into two functional components (1), both of which are required for transcription of tRNA and the adenovirus VA RNA genes. Here we show that these two functional components, designated TFIIIC1 and TFIIIC2, can also be separated by sequence specific DNA affinity chromatography. These results confirm the observation that TFIIIC can be fractionated into two components, which are both required for transcription of VA I and tRNA genes in vitro. Thus in the mammalian reconstituted system, a minimum of three proteins, in addition to RNA polymerase III, are required for the transcription of the VA and tRNA genes in vitro. The DNA binding component, TFIIIC2, binds specifically to the 3' segment of the internal promoter (the B block), demonstrated by its ability to protect this region from digestion by DNase I. TFIIIC2 is the limiting, titratable component in the phosphocellulose C fraction required for the formation of a stable pre-initiation complex on the VAI RNA gene in vitro, as demonstrated with a template competition and rescue assay.
Base Sequence↗
DNA-binding properties and characterization of human transcription factor TFIIIC2.
Interaction between the B-block region of adenovirus VA1 DNA and the human RNA polymerase III transcription factor (TFIII) C2 was analyzed using a gel DNA-binding assay. The retarded band corresponding to the specific complex between TFIIIC2 and the regulatory B-block region was identified by DNase I footprint analysis, competition experiments, and gel shift assays using mutated and truncated virus-associated (VA) 1 DNA probes. The equilibrium constants for the binding reaction with the complete VA1 gene were determined. TFIIIC2 was found to bind to non-specific DNA sequences with a relatively low affinity (equilibrium constant Kn = 6 x 10(4) M-1), and to the B-block sequence with a high affinity (specific constant Ks = 2 x 10(11) M-1). Assuming one site per molecule, the total concentration of binding sites [C0] in the TFIIIC2-containing fractions ranged between 0.6 and 1.6 x 10(-10) M. This corresponded to 1500 TFIIIC2 molecules extracted per 293 cell. Sedimentation analysis of TFIIIC2 on a sucrose gradient showed both VA1 DNA binding activity and in vitro transcription activity cosedimenting with an apparent coefficient of 17-18 S, consistent with a molecular weight of 400-500 kDa. UV cross-linking of a 5-bromo 2'-deoxyuridine-containing, 32P-labeled VA1 probe with the TFIIIC2 fraction, followed by DNase I digestion and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed a single 32P-labeled band migrating as a 250-kDa polypeptide. Compared with the sedimentation data, this result suggests that native TFIIIC2 may be a dimer of the approximately 250-kDa polypeptide.
Adenoviruses, Human↗
Adenovirus proteins from both E1B reading frames are required for transformation of rodent cells by viral infection and DNA transfection.
To determine the requirements for the individual Ad2 E1B proteins during the transformation of rodent cells, viral mutants were constructed with genetic lesions disrupting the coding sequence of either the 175 amino acid residue (175R) or the 495 amino acid residue (495R) E1B proteins. Point mutations generating stop codons very early in the coding sequences were constructed to prevent the expression of amino-terminal protein fragments which might have biological activity. Mutant virus pm1722 contains a point mutation that terminates translation of the 175R protein after three amino acids. It was completely defective for transformation of CREF cells in virion- and DNA-mediated assays. In HeLa cells, pm1722 replicated as well as wild-type virus but produced an extreme cytopathic effect and fragmentation of host-cell DNA. Nonetheless, we provide evidence that the observed transformation defect is not due to the death of transformed cells. The mutant virus dl1520, a double mutant unable to synthesize the 495R protein, was also extremely defective for the transformation of CREF cells in virion- and viral DNA-mediated assays. This result is in contrast to studies with other Ad5 mutants with lesions in the equivalent protein. Possible explanations for this difference are discussed. Replication of dl1520 in HeLa cells was significantly reduced compared to wild-type. Studies with a third mutant virus, pm2022, which contains a stop codon after the second codon of the 495R protein, suggest that very low levels of 495R protein activity are sufficient for a productive infection and significant transforming activity.
Adenovirus Early Proteins↗
Resolution of human transcription factor TFIIIC into two functional components.
tRNA genes and adenovirus viral-associated (VA) genes are transcribed by RNA polymerase III. Transcription of these genes in vitro requires two protein fractions containing transcription factors designated TFIIIB and TFIIIC, in addition to RNA polymerase III. We report that the TFIIIC fraction derived from human cells in culture can be separated into two functional components, which we call TFIIIC1 and TFIIIC2. Both TFIIIC1 and TFIIIC2 fractions are required for in vitro transcription of the VA1 gene. In DNase I "footprinting" experiments, the TFIIIC2 fraction protects the internal control region termed the B block. Addition of the TFIIIC1 fraction extends the footprint over the internal control region called the A block. TFIIIC1 activity is the limiting transcription factor activity required for VA1 transcription in the crude extract. TFIIIC2 activity sediments as a large component of approximately 18 S, while TFIIIC1 activity sediments at approximately 9 S. These data indicate that the two activities are unique components and when added together reconstitute TFIIIC activity.
Adenoviruses, Human↗
Inhibition of host cell RNA polymerase III-mediated transcription by poliovirus: inactivation of specific transcription factors.
The inhibition of transcription by RNA polymerase III in poliovirus-infected cells was studied. Experiments utilizing two different cell lines showed that the initiation step of transcription by RNA polymerase III was impaired by infection of these cells with the virus. The observed inhibition of transcription was not due to shut-off of host cell protein synthesis by poliovirus. Among four distinct components required for accurate transcription in vitro from cloned DNA templates, activities of RNA polymerase III and transcription factor TFIIIA were not significantly affected by virus infection. The activity of transcription factor TFIIIC, the limiting component required for transcription of RNA polymerase III genes, was severely inhibited in infected cells, whereas that of transcription factor TFIIIB was inhibited to a lesser extent. The sequence-specific DNA-binding of TFIIIC to the adenovirus VA1 gene internal promoter, however, was not altered by infection of cells with the virus. We conclude that (i) at least two transcription factors, TFIIIB and TFIIIC, are inhibited by infection of cells with poliovirus, (ii) inactivation of TFIIIC does not involve destruction of its DNA-binding domain, and (iii) sequence-specific DNA binding by TFIIIC may be necessary but is not sufficient for the formation of productive transcription complexes.
Cell Line↗
Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration.
Human cells expressing adenovirus E1A proteins transcribe transfected tRNA and adenovirus VAI genes at greater than 10-fold higher levels than uninfected HeLa cells. Here we show that the increased transcription observed in vivo is reflected in the in vitro transcriptional activity of cell extracts. Depletion of E1A protein from these extracts by immunoprecipitation with a monoclonal antibody did not diminish the activity, suggesting that E1A proteins do not stimulate transcription directly. Fractionation of the extracts by chromatography on phosphocellulose suggests that the higher activity of extracts of adenovirus-infected cells was due to increased activity of the transcription factor (TF) which is the limiting component required for specific initiation of tRNA and VAI transcription in extracts of uninfected HeLa cells, i.e. TFIIIC. Template commitment titrations further suggest that the increased TFIIIC activity was due to an increase in the concentration of active TFIIIC. On the basis of these results and recent genetic analyses of early adenovirus promoters, we suggest that E1A proteins stimulate transcription of adenovirus genes indirectly by increasing the effective in vivo concentration of the limiting cellular transcription factors required for their transcription.
Adenovirus Early Proteins↗
Adenovirus early region 1A protein increases the number of template molecules transcribed in cell-free extracts.
Protein encoded by adenovirus early region 1A (E1A) stimulates transcription from adenovirus promoters in vivo. Here we show that this effect can be observed in vitro. In a run-off transcription assay from the adenovirus serotype 2 (Ad2) major late promoter, extracts prepared 20 hr postinfection were 5-15 times more active than mock-infected-cell extracts prepared in parallel. Similar results were observed for in vitro transcription from the protein IX and E3 adenovirus promoters, whereas a 2-fold increase was observed for the human beta-globin promoter. The increased activities of infected-cell extracts did not depend on the expression of viral late proteins or the small E1A-encoded proteins but did require expression of the large E1A protein. These results are consistent with the large E1A protein stimulating transcription in vitro as it does in vivo. By limiting in vitro transcription to one initiation per template, we found that the higher activity of an infected-cell extract was due to an increase in the number of templates transcribed. These results suggest that the large E1A protein either increases the number of active transcription factors in infected cells or facilitates the interaction of cellular transcription factors with promoter DNA.
Adenovirus Early Proteins↗
Genetic mapping of a major site of phosphorylation in adenovirus type 2 E1A proteins.
Adenovirus early region 1A (E1A) encodes two acidic phosphoproteins which are required for transactivation of viral transcription, efficient viral DNA replication in phase G0-arrested human cells, and oncogenic transformation of rodent cells. Biochemical analysis of in vivo 32P-labeled adenovirus type 2 E1A proteins purified with monoclonal antibodies demonstrated that these proteins were phosphorylated at multiple serine residues. Two-dimensional phosphotryptic peptide maps of wild-type and mutant E1A proteins were used to locate a major site of E1A protein phosphorylation at serine-219 of the large E1A protein. Although this serine fell within a consensus sequence for phosphorylation by the cyclic AMP-dependent protein kinases, experiments with mutant CHO cells defective in these enzymes indicated that it was not. Oligonucleotide-directed mutagenesis was used to substitute an alanine for serine-219. This mutation prevented phosphorylation at this site. Nonetheless, the mutant was indistinguishable from the wild type for early gene transactivation, replication on G0-arrested WI-38 cells, and transformation of cloned rat embryo fibroblast cells.
Adenovirus Early Proteins↗
An immunodominant domain in adenovirus type 2 early region 1A proteins.
Six independent rat hybridoma cell lines producing monoclonal antibodies to human subgroup C adenovirus early region 1A (E1A) proteins were isolated. Competition binding experiments revealed that each of the monoclonal antibodies was directed against the same epitope or overlapping cluster of epitopes on the E1A proteins. Viral E1A deletion mutants and deleted forms of E1A proteins expressed in Escherichia coli were used to localize the antibody recognition sites to sequences between amino acids 23 and 120, encoded within the first exon of the E1A gene. Similarly, polyclonal antisera raised against the trpE-E1A fusion protein, as well as against the native, biologically active E1A protein, were also directed primarily against this immunodominant region.
Adenoviridae↗