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R G Roeder

Publications and source records attributed to R G Roeder.

At least 145 records · Page 8Linked to original sources
PubMed1994

Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers.

The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.

Animals↗
PubMed1993

Identification of human DNA topoisomerase I as a cofactor for activator-dependent transcription by RNA polymerase II.

The transcriptional activation of eukaryotic class II genes by sequence-specific regulatory proteins requires cofactors in addition to the general transcription factors. One cofactor (termed PC3) was purified from HeLa cells and identified by sequence analysis and functional assays as human DNA topoisomerase I (EC5.99.1.2). Under identical conditions PC3 mediates both a net activation of transcription by the acidic activator GAL4-AH and repression of basal transcription, thereby leading to a large induction of transcription by the activator. PC3-mediated activation of transcription is dependent on the presence of both the GAL4-AH activation domain and the TATA-binding protein (TBP)-associated-factors (TAFs) in natural transcription factor TFIID, while repression of basal transcription is observed with either TFIID or the derived TBP alone. These results suggest novel functions, apparently through distinct mechanisms, for human DNA topoisomerase I in the regulation of transcription initiation by RNA polymerase II.

Amino Acid Sequence↗
PubMed1993

Transcriptional activation in yeast by the proline-rich activation domain of human CTF1.

Among eukaryotic transcriptional activators, those with acidic activation domains have been shown to stimulate transcription in organisms ranging from yeast to human. In vivo and in vitro assays have been employed to demonstrate transcriptional enhancement in yeast by the proline-rich activation domain of human CTF1. This result suggests that proline-rich activation domains may, like typical acidic activation domains, functional universally.

CCAAT-Enhancer-Binding Proteins↗
PubMed1993

An alternative pathway for transcription initiation involving TFII-I.

The minimal promoter elements required for initiation by RNA polymerase II include the TATA box and/or an initiator element (Inr) at or near the transcription start site. Studies of the adenovirus major late core promoter (containing both elements) have demonstrated an initiation pathway that involves binding of the transcription factor TFIID (or the derived subunit, the TATA-binding protein TBP (TFIID tau)) to the TATA element, which is facilitated by transcription factor TFIIA, followed by sequential interactions of other general factors. Here we describe a novel pathway that requires an intact Inr and the Inr-binding factor TFII-I (ref. 3). Sequential addition of the general factors generated TFII-I-dependent preinitiation complexes different from those formed with TFIIA. Furthermore, TBP bound cooperatively (with only TFII-I) to an Inr-containing TATA-less promoter, suggesting a means for activation of TATA-less promoters, which nonetheless require TFIID (refs 9-11). These observations provide support for functionally distinct pathways which could be subject to differential regulation by specific activators or repressors.

Adenoviridae↗
PubMed1993

Direct role for Myc in transcription initiation mediated by interactions with TFII-I.

The nuclear proto-oncoprotein Myc has been implicated in the control of cell proliferation and differentiation. Myc participates in transcription and belongs to the basic-helix-loop-helix (bHLH) family of regulatory proteins. Here we show that Myc interacts with TFII-I, a transcription initiation factor that activates core promoters through an initiator element (Inr). As previously observed for the bHLH activator USF, Myc was found to interact cooperatively with TFII-I at both Inr and upstream E-box promoter elements. However, in this case Myc interactions with TFII-I at the Inr lead to an inhibition of transcription initiation. This inhibition is selective for a TFII-I-dependent (as opposed to TFIIA-dependent) initiation pathway and correlates with the prevention of complex formation between the TATA-binding protein TBP (TFIID tau), TFII-I and the promoter. TBP probably interacts with Myc, but only slowly. These observations indicate that Myc has the potential to interact physically and functionally with components of the general transcription machinery.

Adenoviridae↗
PubMed1993

Identification of TFIID components required for transcriptional activation by upstream stimulatory factor.

A TATA box-binding initiation factor, TFIID, plays a central role in the transcriptional regulation by activators. Using anti-TFIID tau (a TATA box-binding component of native TFIID) immunoaffinity chromatography, nine polypeptides (230, 110, 85, 62, 58, 42, 28, 22, and 21 kDa) were identified as native Drosophila TFIID components that are tightly associated with TFIID tau. To verify the functional activity of the purified TFIID complex, template DNA and other transcription factors were reconstituted with purified TFIID bound to the antibody-Sepharose matrix. Immobilized TFIID mediated not only basal transcription but transcriptional activation by upstream stimulatory factor (USF). On the other hand, recombinant TFIID tau immobilized on the same antibody-Sepharose matrix could not mediate activation by USF. These results suggest that one or more of these additional polypeptides are required as functional TFIID subunits for activator-dependent transcription in conjunction with TFIID tau. As further evidence of the relevance of the Drosophila TFIID components identified in this analysis, including the previously unrecognized p230 (Dynlacht, B. D., Hoey, T., and Tjian, R. (1991) Cell 66, 563-576), protein blot analysis showed that TFIID tau interacts specifically and exclusively with p230. This suggests that p230 is an integral subunit of TFIID and that it may play a major role in tethering other subunits to TFIID tau.

Amino Acid Sequence↗
PubMed1993

Transcription factor TFIIB sites important for interaction with promoter-bound TFIID.

Transcription initiation factor TFIIB recruits RNA polymerase II to the promoter subsequent to interaction with a preformed TFIID-promoter complex. The domains of TFIIB required for binding to the TFIID-promoter complex and for transcription initiation have been determined. The carboxyl-terminal two-thirds of TFIIB, which contains two direct repeats and two basic residue repeats, is sufficient for interaction with the TFIID-promoter complex. An extra 84-residue amino-terminal region, with no obvious known structural motifs, is required for basal transcription activity. Basic residues within the second basic repeat of TFIIB are necessary for stable interaction with the TFIID-promoter complex, whereas the basic character of the first basic repeat is not. Functional roles of other potential structural motifs are discussed in light of the present study.

Amino Acid Sequence↗
PubMed1993

Direct binding of yeast transcription factor (TFIID) to the ribosomal protein L32 (rpL32) TATA-less promoter sequence.

The ribosomal protein L32 (rpL32) gene transcribed by RNA polymerase II lacks a canonical TATA element, that binds the transcription factor TFIID tau or TBP (TATA binding protein). Instead this promoter contains an element, termed gamma, located at -30 relative to the transcription initiation site. We previously reported that, despite the lack of a canonical TATA element the rpL32 gene utilizes yeast TFIID tau for its transcriptional initiation. Whether TFIID tau participates in rpL32 gene transcription by binding directly to a promoter element or through another protein has not been resolved. These studies reveal that proteins ranging in size from 20-40 kDa binds to the gamma-element. The 40 kDa protein(s) displays strong affinity for the canonical TATA element and may be related or equivalent to TFIID tau. Furthermore, cloned and purified yeast TFIID (TBP) binds directly to the gamma-element implying that the gamma-element directs RNA polymerase II-dependent transcription of the rpL32 gene.

Base Sequence↗
PubMed1993

The Drosophila 110-kDa transcription factor TFIID subunit directly interacts with the N-terminal region of the 230-kDa subunit.

Transcription initiation factor TFIID is a multimeric protein complex that plays a central role in transcriptional regulation by facilitating promoter responses to various activators. cDNAs encoding the 110-kDa subunit of Drosophila TFIID (p110) were isolated with a degenerate oligodeoxynucleotide probe based on an amino acid sequence of the purified protein. The entire cDNA sequence contains an open reading frame encoding a 921-amino acid polypeptide with a calculated molecular mass of 99,337 Da. The recombinant protein expressed in Sf9 cells via a baculovirus vector interacts directly with the 230-kDa subunit of TFIID (p230). Together with the previous observation that the TATA box-binding subunit of TFIID (TFIID tau or TBP) interacts directly with only p230 among the TFIID subunits, this result suggests that p110 forms a complex with TFIID tau via p230. A binding study using various p230 mutants indicated that both p110 and TFIID tau interact with the N-terminal 352-amino acid portion of p230, suggesting a functional communication between p110 and TFIID tau via p230 interactions.

Amino Acid Sequence↗
PubMed1993

Functional dissection of TFIIB domains required for TFIIB-TFIID-promoter complex formation and basal transcription activity.

The protein TFIIB is a general transcription initiation factor that interacts with a promoter complex (D.DNA) containing the TATA-binding subunit (TFIID tau, or TBP) of TFIID to facilitate subsequent interaction with RNA polymerase II (ref. 2) through the associated TFIIF (ref. 3). The potential bridging function of TFIIB raises the possibility of two structural domains and emphasizes the importance of TFIIB structure-function studies for a further understanding of preinitiation complex assembly and function. Here we show that human TFIIB (refs 5,6) is comprised of functionally distinct N- and C-terminal domains. The C-terminal domain, containing the direct repeats and associated basic regions, is necessary and sufficient for interaction with the D.DNA complex. By contrast, the N-terminal domain that is dispensable for formation of the TFIID tau-TFIIB-promoter (D.B.DNA) complex is required for subsequent events leading to basal transcription initiation. On the basis of these results, we discuss structural and functional similarities between TFIIB and TFIID tau, which have similar structural organization and motifs.

Binding Sites↗
PubMed1993

The p250 subunit of native TATA box-binding factor TFIID is the cell-cycle regulatory protein CCG1.

The protein TFIID is a general transcription factor which initiates preinitiation complex assembly through direct interaction with the TATA promoter element. It is a multisubunit complex containing a small TATA-binding polypeptide (TBP) and other TBP-associated factors (TAFs) ranging in size from about 30-250K (refs 7-10). Although native TFIID can mediate both activator-independent (basal) and activator-dependent transcription in reconstituted systems, TBP itself can mediate only basal transcription, even in cases where TBP or the general factor TFIIB are known to interact directly with transcriptional activators. TFIID subunits other than TBP must therefore be essential cofactors, and thus potential targets for activators, consistent with earlier demonstrations that activators interact with TFIID (refs 3, 5, 16, 17). Here we show that the 250K subunit of TFIID is identical to a gene product previously implicated in progression through the late G1 phase of the cell cycle. Part of p250 may thus serve a specific function in the activation of a subset of genes important for cell cycle progression.

Amino Acid Sequence↗
PubMed1993

Genetic and biochemical analyses of yeast TATA-binding protein mutants.

We have taken a combined genetic and biochemical approach to study TATA-binding protein (TBP) structure-function relationships. Using site-directed mutagenesis coupled with a screen for conditional lethal growth, we have isolated a number of temperature-sensitive TBP alleles in the region of amino acid positions 188, 189, and 190. Conditional growth is not a result of increased TBP turnover as most of the mutant proteins are stable in vivo as evidenced by immunoblot detection of TBP steady-state levels. DNA binding assays reveal that mutations at position 188 do not affect DNA binding activity of these mutants, even at high temperatures. Utilizing whole cell extracts which contain mutant TBPs in in vitro transcription experiments, we confirm that TBP is required for transcription by all three nuclear polymerases. However, certain of our TBP mutants are only compromised for RNA polymerase II transcription.

Alleles↗
PubMed1993

Human transcription factor USF stimulates transcription through the initiator elements of the HIV-1 and the Ad-ML promoters.

Earlier in vitro studies identified USF as a cellular factor which activates the adenovirus major late (Ad-ML) promoter by binding to an E-box motif located at position -60 with respect to the cap site. Purified USF contains 44 and 43 kDa polypeptides, and the latter was found (by cDNA cloning) to be a helix-loop-helix protein. In this report, we demonstrate a 25-to 30-fold stimulation of transcription via an upstream binding site by ectopic expression of the 43 kDa form of USF (USF43) in transient transfection assays. More recent data have also revealed alternate interactions of USF43 at pyrimidine-rich (consensus YYAYTCYY) initiator (Inr) elements present in a variety of core promoters. In agreement with this observation, we show here that USF43 can recognize the initiator elements of the HIV-1 promoter, as well as those in the Ad-ML promoter, and that ectopic expression of USF43 can stimulate markedly the corresponding core promoters (TATA and initiator elements) when analyzed in transient co-transfection assays. Mutations in either Inr 1 or Inr 2 reduced the USF43-dependent transcription activity in vivo. In addition, in vitro transcription assays showed that mutations in either or both of the Inr 1 and Inr 2 sequences of the HIV-1 and Ad-ML promoters could affect transcription efficiency, but not the position of the transcriptional start site. These results indicate that USF43 can stimulate transcription through initiator elements in two viral promoters, although the exact mechanism and physiological significance of this effect remain unclear.

Base Sequence↗
PubMed1993

Unique TATA-binding protein-containing complexes and cofactors involved in transcription by RNA polymerases II and III.

Two multisubunit complexes containing the TATA-binding protein (TBP) were isolated from HeLa cells constitutively expressing the FLAG epitope-tagged TBP using antibody affinity and peptide elution methods. One of the complexes (f:TFIID), isolated from the P11 0.85 M KCl fraction, contains at least 13 specific TBP-associated factors (TAFs) and can mediate activator-dependent transcription by RNA polymerase II. Importantly, activator function through the highly purified f:TFIID complex still requires a general cofactor fraction containing upstream factor stimulatory activity (USA). As previously observed with partially purified activator-competent natural TFIID, f:TFIID generates extended TATA-dependent footprints on the intrinsically strong adenovirus major late promoter (MLP) but only restricted footprints on the weak adenovirus E1b and E4 and HIV (core) promoters. Along with previous demonstrations of activator-induced downstream TFIID interactions on the E4 promoter, these results argue for a relationship between downstream interactions and overall promoter strength. Initiator-like sequences appear not to be essential for downstream interactions since they have no effect on downstream MLP interactions when mutated, do not effect downstream interactions on the HIV promoter and are not present on the inducible E4 promoter. The other multisubunit complex (f:TFIIIB), isolated from the P11 0.30 M KCl fraction, contains four specific TAFs and can substitute for one of the fractions (TFIIIB) required for RNA polymerase III (pol III) transcription. Neither f:TFIID nor TBP could substitute for this pol III TBP-containing fraction. This plus the fact that f:TFIIIB failed to generate a footprint on the MLP underscores the importance of TAFs in determining promoter specificity by different RNA polymerases.

Adenovirus E1B Proteins↗
PubMed1993

A single cDNA, hTFIIA/alpha, encodes both the p35 and p19 subunits of human TFIIA.

TFIIA is a transcription factor that, by interacting with the TATA-binding subunit (TBP) of TFIID, modulates transcription initiation by RNA polymerase II in vitro. By use of a mobility shift assay, TFIIA was purified from HeLa cells as a complex of 35-, 19-, and 12-kD subunits. Oligonucleotides were used to isolate a human cDNA clone, hTFIIA/alpha, which encodes a 55-kD protein with homology to the product of the yeast gene TOA1. The open reading frame of hTFIIA/alpha contains peptide sequences obtained from both the p35 and p19 subunits of natural human TFIIA, and thus encodes these two subunits. Consistent with this, antiserum raised against the 55-kD hTFIIA/alpha-encoded protein reacted with both the p35 and p19 subunits of natural TFIIA, and the recombinant protein could functionally replace those subunits in a mobility shift assay with renatured p12. An efficient affinity purification for natural human TFIIA was suggested by the sequence of the hTFIIA/alpha protein and demonstrated biochemically. Finally, transcription from the adenovirus major late promoter was greatly reduced in nuclear extracts depleted with anti-TFIIA/alpha serum and was restored to original levels by the readdition of purified human TFIIA.

Amino Acid Sequence↗
PubMed1993

Drosophila 230-kD TFIID subunit, a functional homolog of the human cell cycle gene product, negatively regulates DNA binding of the TATA box-binding subunit of TFIID.

A Drosophila cDNA encoding the largest TFIID subunit (p230) was isolated using a degenerate oligodeoxynucleotide probe based on an amino acid sequence of the purified protein. The entire cDNA sequence contains an open reading frame encoding a polypeptide of 2068 amino acids, corresponding to a calculated molecular mass of 232 kD. The deduced amino acid sequence showed a strong sequence similarity with the protein encoded by a human gene (CCG1) implicated in cell cycle progression through G1, suggesting that p230 may be a target for cell cycle regulatory factors. The recombinant protein expressed in Sf9 cells via a baculovirus vector interacts directly with the TATA box-binding subunit of TFIID (TFIID tau or TBP) from Drosophila, human, and yeast. Surprisingly, recombinant p230 inhibits the TATA box-binding activity and function of TFIID tau, suggesting that p230 interactions with TFIID tau, and possible modulations thereof by other factors may play an important role in TFIID function.

Amino Acid Sequence↗
PubMed1993

Unliganded thyroid hormone receptor inhibits formation of a functional preinitiation complex: implications for active repression.

The thyroid hormone receptor (TR) belongs to the steroid/nuclear receptor superfamily of ligand-inducible transcription factors. Numerous studies using transient transfection assays have demonstrated that in the absence of thyroid hormone (T3), unliganded TR acts as a constitutive repressor of transcription on genes bearing TR-response elements. We examined the molecular mechanism of TR repression in vitro using both HeLa nuclear extracts and purified basal factors. Here, we show that unliganded TR is an active transcriptional repressor, distinct from passive repressors that compete with activators for DNA binding. Repression by TR can be relieved by adding the T3 analog triiodothyroactic acid, suggesting that liganded TR undergoes a conformational change that masks or disrupts the repressor function. Repression by TR is mediated through the basal transcription machinery and can occur independently of previously characterized TATA-binding protein-associated cofactors thought to be involved in either basal repression or activator-dependent transcription. TR inhibits transcription at an early step during preinitiation complex (PIC) assembly, as preassembled PICs are refractory to the inhibitory effects of TR.

Animals↗