PubMed Health⌕ Search

Biomedical subjects

D Reinberg

Publications and source records attributed to D Reinberg.

At least 109 records · Page 6Linked to original sources
PubMed1991

The small subunit of transcription factor IIF recruits RNA polymerase II into the preinitiation complex.

We found that transcription factor IIF mediates the association of RNA polymerase II with promoter sequences containing transcription factors IID, IIB, and IIA (DAB complex). The resulting DNA-protein complex contained RNA polymerase II and the two subunits of transcription factor IIF (RAP 30 and RAP 74). Cloned human RAP 30 was sufficient for the recruitment of RNA polymerase II to the DAB complex. This ability of RAP 30 to recruit RNA polymerase to a promoter is also a characteristic of sigma factors in prokaryotes.

Adenoviridae↗
PubMed1991

Binding of general transcription factor TFIIB to an acidic activating region.

A central issue in eukaryotic transcriptional regulation is the mechanism by which promoter-specific transcription factors (activators) stimulate transcription. Two lines of evidence indicate that the general transcription factor TFIIB is a pivotal component in the mechanism by which an acidic activator functions. First, during assembly of the preinitiation complex TFIIB binding is a rate-limiting step enhanced by an acidic activator. Second, the TFIIB activity in a HeLa cell nuclear extract is specifically retained on a column containing an acidic activating region. But because our previous study monitored only TFIIB activity, it remains possible that the interaction between TFIIB and the acidic activating region is mediated through additional proteins, for example, those designated as adaptors, coactivators or mediators. A complementary clone encoding TFIIB has recently been isolated and shown to encode a polypeptide of relative molecular mass 35,000. Here we report that TFIIB expressed in and purified from Escherichia coli (recombinant TFIIB) binds directly to the potent acidic activating region of the herpes simplex virus-1 VP16 protein.

Base Sequence↗
PubMed1991

The initiator directs the assembly of a transcription factor IID-dependent transcription complex.

Highly purified RNA polymerase II was found to be able to weakly recognize the initiator (Inr) present in the adenovirus IVa2 and major late promoters. The association of RNA polymerase II with the Inr was enhanced by the general transcription factors. The Inr was capable of directing the formation of a DNA-protein complex. Transcription competent complexes on the adenovirus major late and IVa2 promoters appear to be formed by alternative pathways mediated through the Inr and/or "TATA" motif. The presence of both motifs, however, is required for efficient transcription utilizing a discrete start site. Complexes formed at either site required transcription factor TFIID, the TATA binding protein. Consistent with this observation, a TFIID requirement was demonstrated for transcription from a mutant adenovirus major late promoter construct lacking a functional TATA motif.

Adenoviridae↗
PubMed1991

Cloning of a human gene encoding the general transcription initiation factor IIB.

Transcription factor IIB (TFIIB) has a central role in transcription of class II genes. The purification of the human TFIIB protein and isolation of a complementary DNA encoding TFIIB activity is reported here. The sequence of TFIIB, which seems to be encoded by a single gene, contains a repeated motif, in addition to a motif with similarity to the prokaryotic sigma-factors. The recombinant protein expressed in bacteria substituted for all the functions attributed to the human TFIIB protein.

Amino Acid Sequence↗
PubMed1991

Direct interaction between adenovirus E1A protein and the TATA box binding transcription factor IID.

Adenovirus E1A has long been known to activate/repress cellular and viral transcription. The transcriptional activity of nuclear extracts was depleted after chromatography on immobilized E1A protein columns that specifically retained the transcription factor (TF) IID. Stronger direct interactions between E1A and human TFIID than between E1A and yeast TFIID suggest that the unique sequences of the human protein may be involved. We have demonstrated that this interaction occurs directly between bacterially produced E1A and bacterially produced human TFIID in a protein blot assay. We propose that E1A protein may transduce regulatory signals from upstream activators to basal elements of the transcriptional machinery by contacting TFIID.

Adenovirus Early Proteins↗
PubMed1991

Interaction of CAP sequence site binding factor and transcription factor IID preceding and following binding to the adenovirus 2 major late promoter.

Interaction of cloned yeast, drosophila, and human transcription factor IID (yTFIID, dTFIID, and hTFIID, respectively) with the adenovirus 2 major late promoter (Ad2 MLP) confers a more limited pattern of DNase I protection than that obtained using highly purified native hTFIID (Hahn, S., Buratowski, S., Sharp, P. A. and Guarente, L. (1989) EMBO J. 8, 3379-3382; Van Dyke, M. W., and Sawadogo, M. (1990) Mol. Cell. Biol. 10, 3415-3420; Horikoshi, M., Wang, C.K., Fujii, H., Cromlish, J.A., Weil, P.A., and Roeder, R.G. (1989) Nature 341, 299-303; Peterson, M. G., Tanese, N., Pugh, B.F., and Tjian, R. (1990) Science 248, 1625-1630; Hoey, T., Dynlacht, B. D., Peterson, M.G., Pugh, B.F., and Tjian, R. (1990) Cell 61, 1179-1186). Since the mass of the cloned TFIIDs is considerably less than that of native hTFIID (27-38 kDa versus 120-140 kDa), it is considered likely that native hTFIID exists as a mixed heterodimer. We have recently identified, purified, and characterized a novel transcription factor that binds to the CAP site region (+1 to +23) of the Ad2 MLP. This CAP site binding factor, designated CBF, is required for optimal transcriptional activity. We now show that when bound to the Ad2 MLP, yTFIID and CBF interact to generate the extended pattern of DNase I protection conferred by native hTFIID. In addition, bound yTFIID and CBF interact such that the stability of the complex exceeds that of each factor bound alone. We also demonstrate the existence in nuclear extracts of a hTFIID and CBF heterodimer by the electrophoretic mobility shift analysis. CBF, therefore, may represent the first identified member of a large family of gene-specific TFIID-associated factors that are required for the regulated gene-specific expression of TFIID activity.

Adenoviruses, Human↗
PubMed1991

Factors involved in specific transcription by mammalian RNA polymerase II. Purification and functional analysis of general transcription factor IIE.

Mammalian RNA polymerase II transcription factor IIE (TFIIE) was purified to apparent homogeneity. The activity copurified with polypeptides of 34 and 56 kDa. The 56-kDa subunit was sufficient for low levels of transcription activity in a transcription system reconstituted in vitro with highly purified general transcription factors and RNA polymerase II. The 34-kDa polypeptide was found to be stimulatory. The native molecular mass of TFIIE, as determined by gel filtration was estimated to be approximately 200 kDa, suggesting that TFIIE exists in solution as a tetramer composed of two 56-kDa and two 34-kDa polypeptides. Consistent with previous studies demonstrating an interaction of TFIIE with RNA polymerase II, we found that the entry of TFIIE into the transcription cycle was subsequent to the entry of RNA polymerase II.

Deoxyribonucleoproteins↗
PubMed1991

Role of the mammalian transcription factors IIF, IIS, and IIX during elongation by RNA polymerase II.

We have used a recently developed system that allows the isolation of complexes competent for RNA polymerase II elongation (E. Bengal, A. Goldring, and Y. Aloni, J. Biol. Chem. 264:18926-18932, 1989). Pulse-labeled transcription complexes were formed at the adenovirus major late promoter with use of HeLa cell extracts. Elongation-competent complexes were purified from most of the proteins present in the extract, as well as from loosely bound elongation factors, by high-salt gel filtration chromatography. We found that under these conditions the nascent RNA was displaced from the DNA during elongation. These column-purified complexes were used to analyze the activities of different transcription factors during elongation by RNA polymerase II. We found that transcription factor IIS (TFIIS), TFIIF, and TFIIX affected the efficiency of elongation through the adenovirus major late promoter attenuation site and a synthetic attenuation site composed of eight T residues. These factors have distinct activities that depend on whether they are added before RNA polymerase has reached the attenuation site or at the time when the polymerase is pausing at the attenuation site. TFIIS was found to have antiattenuation activity, while TFIIF and TFIIX stimulated the rate of elongation. In comparison with TFIIF, TFIIS is loosely bound to the elongation complex. We also found that the activities of the factors are dependent on the nature of the attenuator. These results indicate that at least three factors play a major role during elongation by RNA polymerase II.

Adenoviruses, Human↗
PubMed1990

Factors involved in specific transcription by mammalian RNA polymerase II. Purification and subunit composition of transcription factor IIF.

Transcription factor IIF (TFIIF), a protein factor required for transcription of class II genes, has been purified to near homogeneity. TFIIF is a heterodimer of 30- and 78-kDa polypeptides. Both the 30- and 78-kDa polypeptides were necessary to reconstitute TFIIF activity. TFIIF activity eluted from a gel filtration column with an apparent molecular mass of 220 kDa, suggesting that the native factor exists as a dimer of the heterodimer.

Animals↗
PubMed1990

A TATA-like sequence located downstream of the transcription initiation site is required for expression of an RNA polymerase II transcribed gene.

TFIID, the TATA-binding protein, was found to stimulate transcription from the adenovirus IVa2 promoter, a promoter considered to lack the TATA motif. Remarkably, a TATA-like sequence element located downstream of the transcription start site binds TFIID and is required for TFIID-dependent transcription from the IVa2 promoter. Transcription from the IVa2 and the adjacent adenovirus major late promoter (Ad-MLP) is divergent, and the cap sites are separated by 212 nucleotides. Nevertheless, the TATA motifs of the IVa2 promoter and Ad-MLP were found to be oriented in the same direction. An initiator motif around the transcription start site is located in the IVa2 promoter, and in contrast to the TATA motifs, the IVa2-initiator is in the opposite orientation with respect to the initiator of the Ad-MLP. A model is presented in which the polar nature of the initiator governs the direction of transcription. We propose that RNA polymerase II and accessory factors recognize the initiator in an orientation-dependent fashion. The recognition of the IVa2 initiator by RNA polymerase is enhanced by the binding of TFIID to the downstream TATA motif.

Adenoviridae↗
PubMed1990

Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex.

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.

Base Sequence↗
PubMed1989

TPA can overcome the requirement for EIa and together act synergistically in stimulating expression of the adenovirus EIII promoter.

We have examined the control of gene expression from the adenovirus early region III (Ad-EIII) promoter, which contains two previously defined elements, the AP1 and ATF sites. We found that the AP1 element is capable of mediating activation by the adenovirus immediate early (EIa) gene products. Consistent with studies demonstrating that the AP1 site mediates signal transduction in response to 12-O-tetradecanoylphorbol 13-acetate (TPA) we have shown that TPA can activate Ad-EIII expression and overcome the requirement for EIa. Together TPA and EIa elicited a synergistic response in expression from the Ad-EIII promoter during both transient expression assays and viral infections. This synergistic effect required the AP1 element. An EIII promoter construct, in which sequences upstream of the TATA box had been replaced with four AP1 sites, was responsive to TPA and EIa and in combination promoted the synergistic effect. The analysis of specific factors involved in transcription from the Ad-EIII indicated that proteins recognizing the ATF and AP1 sites were important in expression from this promoter in vitro. Purification of protein factors that specifically stimulated EIII expression resulted in the isolation of a set of factors of the AP1 family. Affinity purified AP1 recognized and activated transcription through both the AP1 and ATF elements. In addition, a protein fraction was identified with DNA binding activity specific for the ATF element. This fraction was dependent on the ATF site for transcriptional activity.

Adenovirus Early Proteins↗
PubMed1989

Phosphorylation of cellular proteins regulates their binding to the cAMP response element.

We have studied the protein factors that promote transcription via binding to the cAMP response element (CRE) present in the adenovirus early region III (EIII) and early region IV (EIV) promoters. Three sets of CRE-binding phosphoproteins, ranging in molecular mass from 65-72, 38-43, and 31-37 kDa, were identified in vivo from HeLa cells. Western blot analysis revealed that all three sets of proteins identified were immunologically related to the transcription factor AP1. We found that binding of these proteins to the CRE could be regulated by phosphorylation in vitro. EivF, a 65-72-kDa protein was found to bind specifically to the adenovirus EIV promoter. We have also shown that the smaller molecular mass proteins of 31-37 and 38-43 kDa were able to bind to the CRE present in the adenovirus EIV promoter, as well as to two related DNA elements present in the adenovirus EIII promoter, the ATF and AP1 sites. Phosphorylation of these proteins with the cAMP-dependent protein kinase, affected their transcriptional activity and binding affinity to the three sites. Furthermore, the binding specificity of the 31-37-kDa polypeptides was mediated by cAMP-dependent protein kinase in vitro. Our data suggests that phosphorylation of factors that bind to the CRE may, in part, underlie the cellular response to the adenovirus-encoded Ela protein.

Adenoviridae↗
PubMed1989

The adenovirus inverted terminal repeat functions as an enhancer in a cell-free system.

Two binding sites for EivF, a factor involved in transcription from the adenovirus early promoter iv (Eiv), were mapped within the adenovirus inverted terminal repeats (ITR). Consistent with the observation that EivF was required to initiate transcription from the Eiv promoter and with the demonstration that two EivF binding sites were present in the ITR, we show that the inverted terminal repeat region was able to promote transcription from the CAP site of the Eiv promoter in vitro and in an EIa-dependent fashion in vivo. The minimum sequence within the ITR capable of directing EIa-dependent transcription consists of forty nine nucleotides comprising two EivF binding sites and at least one Sp1 binding site. This 49-base pair fragment possesses the characteristics of an enhancer which is induced by EIa. The enhancer is active in HeLa cell nuclear extracts. Transcription directed by the ITR required EivF and the general transcription factors. The addition of purified Sp1 factor specifically stimulated transcription which correlates with the presence of Sp1 binding sites between the two EivF recognition sites.

Adenoviridae↗
PubMed1989

Factors involved in specific transcription by mammalian RNA polymerase II. Factors IIE and IIF independently interact with RNA polymerase II.

The purification and characterization of transcription factor IIF (TFIIF), a factor required for transcription by the RNA polymerase II machinery, is described. TFIIF was isolated from the previously described IIE protein fraction. TFIIF enters into the transcription cycle via a preinitiation complex, and it is required for the formation of a complex capable of initiating transcription in the presence of heparin concentrations that inhibit the action of a free factor. TFIIF and TFIIE independently interacted with purified RNA polymerase II. TFIIF and TFIIE were both required for transcription of several class II promoters, including a promoter that lacks the conserved TATA box. Interestingly TFIIF was absolutely required for the formation of a preinitiation complex; however, it also affected the elongation phase of the transcription cycle. TFIIF, together with the previously described elongation factor TFIIS, was required for efficient elongation.

Adenoviruses, Human↗
PubMed1989

Factors involved in specific transcription by mammalian RNA polymerase II. Role of factors IID and MLTF in transcription from the adenovirus major late and IVa2 promoters.

The role of the adenovirus major late upstream transcription factor (MLTF) in transcription from the adenovirus major late and the IVa2 promoters was studied. The transcription initiation site of the IVa2 promoter is located 210 nucleotides upstream from the CAP site of the major late promoter. Transcription from these two promoters occurs on different DNA strands. Thus, this divergent transcription suggests that the same factor could simultaneously regulate the expression of two different genes. This was investigated utilizing a reconstituted transcription system in vitro. The addition of MLTF to reaction mixtures containing the purified general transcription factors and the major late promoter resulted in a 10-12-fold stimulation of transcription. This stimulation was because of an increase of the stability of the preinitiation complex. MLTF allowed DNA template molecules to undergo multiple rounds of transcription. MLTF also stimulated transcription from the adenovirus-encoded IVa2 promoter. Surprisingly, reconstitution experiments indicated that transcription from the IVa2 promoter which does not have a TATA sequence required all the previously described general transcription factors, including TFIID, the TATA binding protein. The requirement for TFIID was demonstrated by reconstitution experiments as well as by oligonucleotide competition experiments. The implications of this observation are discussed.

Adenoviruses, Human↗
PubMed1988

Factors involved in specific transcription by mammalian RNA polymerase II. RNA polymerase II-associating protein 30 is an essential component of transcription factor IIF.

Transcription from class II promoters requires five general factors, IIA, IIB, IID, IIE, and IIF, in addition to RNA polymerase II for basal levels of transcription (Reinberg, D., Flores, O., and Buckbinder, L. (1987) in Molecular Biology of RNA: New Perspectives (Inouye, M., and Dudock, B., eds) pp. 423-439, Academic Press, Orlando, FL). A protein fraction containing transcription factors (TF) IIE and IIF was able to reconstitute transcription from the adenovirus major late promoter when added to extracts depleted of the RNA polymerase II-associating proteins RAP 30 and RAP 74 (Sopta, M., Carthew, R.W., and Greenblatt, J. (1985) J. Biol. Chem. 260, 10353-10360). Studies with monoaffinity-purified antibodies directed against RAP 30 demonstrated, by Western blot analysis, that RAP 30 copurifies on five columns with transcription factor IIF. That RAP 30 is a functional component of TFIIF was also demonstrated; preincubation of anti-RAP 30 antibodies with purified TFIIF inhibited transcription. Inhibition of transcription was overcome by the addition of purified TFIIF. RAP 30 is an integral part of a preinitiation complex; the incubation of all the general transcription factors with a promoter-containing DNA, prior to the addition of the anti-RAP 30 antibodies, resulted in the formation of a DNA-protein complex that was not inhibited by the antibodies. Incubation of the transcription factors in the absence of a promoter-containing DNA resulted in a complex that was partially resistant to the antibodies.

Adenoviruses, Human↗