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R G Roeder

Publications and source records attributed to R G Roeder.

At least 163 records · Page 9Linked to original sources
PubMed1993

Potential RNA polymerase II-induced interactions of transcription factor TFIIB.

The ubiquitous transcription factor TFIIB is required for initiation by RNA polymerase II and serves as a target of some regulatory factors. The carboxy-terminal portion of TFIIB contains a large imperfect direct repeat reminiscent of the structural organization of the TATA-binding component (TBP) of TFIID, as well as sequence homology to conserved regions of bacterial sigma factors. The present study shows that the carboxy-terminal portion of TFIIB, like that of TBP, is folded into a compact protease-resistant core. The TFIIB core, unlike the TBP core, is inactive in transcription but retains structural features that enable it to form a complex with promoter-bound TFIID. The protease-susceptible amino terminus appears to contain components responsible for direct interaction with RNA polymerase II (in association with TFIIF) either on the promoter (in association with TFIID) or independently. In addition, core TFIIB (but not intact TFIIB) extends the footprint of TBP on promoter DNA, suggesting that TFIIB has a cryptic DNA-binding potential. These results are consistent with a model in which TFIIB, in a manner functionally analogous to that of bacterial sigma factors, undergoes an RNA polymerase II-dependent conformational change with resultant DNA interactions during the pathway leading to a functional preinitiation complex.

Amino Acids↗
PubMed1993

Molecular cloning, expression, and characterization of the Drosophila 85-kilodalton TFIID subunit.

Transcription initiation factor TFIID is a multimeric protein complex that plays a central role in mediating promoter responses to various activators and repressors. To further understand the role of the 85-kDa TFIID subunit (p85), we have cloned the corresponding cDNA with a probe based on an amino acid sequence of the purified protein. The recombinant p85 interacts directly with both the TATA box-binding subunit (TFIID tau or TBP) and the 110-kDa subunit (p110) of TFIID, suggesting that p85 may play a role in helping to anchor p110 within the TFIID complex and, with other studies, that TFIID assembly and function may involve a concerted series of subunit interactions. Interestingly, the carboxy terminus of p85 contains eight of the WD-40 repeats found originally in the beta subunit of G proteins and more recently in other transcriptional regulatory factors. However, truncated p85 lacking all the WD-40 repeats maintained interactions with both TFIID tau and p110. These observations leave open the possibility of a distinct function for the WD-40 repeats, possibly in transducing signals by interactions with transcriptional regulators and/or other components of the basic transcriptional machinery.

Amino Acid Sequence↗
PubMed1992

Identification of human TFIID components and direct interaction between a 250-kDa polypeptide and the TATA box-binding protein (TFIID tau).

Previous studies have indicated that human transcription initiation factor TFIID is a large complex that contains a TATA-binding polypeptide (TFIID tau or TBP) and other components that qualitatively alter promoter interactions and are uniquely required for activator-dependent (versus basal) transcription. TFIID tau-specific antibody columns have been employed to identify a number of human TFIID polypeptides that are tightly associated with TFIID tau. These differ in size from polypeptides in known general initiation factors, including the initiator-binding factor (TFII-I) which shares some promoter binding characteristics with TFIID. The largest component (p250) identified in TFIID was shown to interact directly and tightly with TFIID tau, suggesting that it may play a major role in the assembly of the TFIID complex.

DNA-Binding Proteins↗
PubMed1992

Purification and characterization of two forms of human transcription factor IIIC.

We showed previously that HeLa cell nuclear extracts contain two forms of transcription factor IIIC (TFIIIC) that formed chromatographically distinct TFIIIC-promoter complexes (Hoeffler, W. K., Kovelman, R., and Roeder, R. G. (1988) Cell 53, 907-920). One of these forms, the upper-band form, correlated with TFIIIC transcriptional activity, whereas the lower-band form bound to the VA1 promoter but supported little or no transcriptional activity. Using both transcription and DNA-binding assays, we have now purified both the upper-band form and the lower-band form of TFIIIC to near-homogeneity. The upper-band form is composed of five polypeptides with estimated sizes of 220, 110, 102, 90, and 63 kDa. The largest of these polypeptides can be cross-linked to the VA1 promoter. The lower-band form has a polypeptide structure similar to that of the upper-band form except for the absence or modification of the 110-kDa subunit. Direct assays show that the lower-band form is indeed transcriptionally inactive at all stages of purification, even when assayed with an unfractionated, heat-treated nuclear extract as a complementation system. This inactivity does not result from altered DNA-binding properties; instead, we suggest that the alteration of one of the subunits of TFIIIC renders it unable to interact productively with a downstream component of the transcription complex.

Base Sequence↗
PubMed1992

The helix-loop-helix/leucine repeat transcription factor USF can be functionally regulated in a redox-dependent manner.

We showed previously that the DNA-binding capacity of the helix-loop-helix/leucine repeat transcription factor USF43 is lowered dramatically under nonreducing conditions. This report defines the molecular basis of this effect by showing (i) that the only two USF43 cysteine residues, both present within the helix-loop-helix protein-protein interface domain, are required for this regulation, (ii) that the sulfhydryl groups of these cysteine residues are the actual targets of this regulation, (iii) that oxidation of these groups results in both intra- and intermolecular nonrandom covalent links, (iv) that this redox modulation of USF43 DNA-binding potential can translate in vitro into a specific modulation of its ability to activate transcription from a USF-responsive promoter. The implications of these modulations of USF43 function in response to redox changes are discussed with regard to the apparent paradox of USF strong activation potential and its ubiquitous distribution in all cell types tested.

Amino Acid Sequence↗
PubMed1992

Crystal structure of TFIID TATA-box binding protein.

The structure of a central component of the eukaryotic transcriptional apparatus, a TATA-box binding protein (TBP or TFIID tau) from Arabidopsis thaliana, has been determined by X-ray crystallography at 2.6 A resolution. This highly symmetric alpha/beta structure contains a new DNA-binding fold, resembling a molecular 'saddle' that sits astride the DNA. The DNA-binding surface is a curved, antiparallel beta-sheet. When bound to DNA, the convex surface of the saddle would be presented for interaction with other transcription initiation factors and regulatory proteins.

Amino Acid Sequence↗
PubMed1992

A novel B cell-derived coactivator potentiates the activation of immunoglobulin promoters by octamer-binding transcription factors.

A novel B cell-restricted activity, required for high levels of octamer/Oct-dependent transcription from an immunoglobulin heavy chain (IgH) promoter, was detected in an in vitro system consisting of HeLa cell-derived extracts complemented with fractionated B cell nuclear proteins. The factor responsible for this activity was designated Oct coactivator from B cells (OCA-B). OCA-B stimulates the transcription from an IgH promoter in conjunction with either Oct-1 or Oct-2 but shows no significant effect on the octamer/Oct-dependent transcription of the ubiquitously expressed histone H2B promoter and the transcription of USF- and Sp1-regulated promoters. Taken together, our results suggest that OCA-B is a tissue-, promoter-, and factor-specific coactivator and that OCA-B may be a major determinant for B cell-specific activation of immunoglobulin promoters. In light of the evidence showing physical and functional interactions between Oct factors and OCA-B, we propose a mechanism of action for OCA-B and discuss the implications of OCA-B for the transcriptional regulation of other tissue-specific promoters.

B-Lymphocytes↗
PubMed1992

Yeast transcription factor IID participates in cell-free transcription of a mammalian ribosomal protein TATA-less promoter.

We analysed transcription of the gene for the ribosomal protein (rp) L32 of the mouse, which is transcribed in mouse L1210 nuclear extracts in vitro. The rpL32 gene lacks a canonical TATA box. Hence it has been suggested that this gene has an alternative transcription pathway not requiring transcription factor IID (TFIID). Selective inactivation of TFIID in nuclear extract completely abolished the transcription of rpL32 in vitro. Selective inactivation was restored by the addition of cloned and purified yeast TFIID (yTFIID), indicating that this TATA-less rpL32 promoter utilizes TFIID for its transcription initiation. Furthermore, addition of an oligonucleotide-containing TATA sequence interfered with the rpL32 transcription and this was overcome by the addition of yTFIID. To further examine the stage of involvement of TFIID in rpL32 transcription, TATA oligonucleotide was added to nuclear extract before and after the formation of the transcription complex. The results reveal that TFIID associates with the pre-initiation complex and that this complex is largely resistant to added TATA oligonucleotide. Our results show, for the first time, that the TATA-less rpL32 gene utilizes TFIID for transcription initiation.

Animals↗
PubMed1992

Isolation and characterization of a cDNA encoding Drosophila transcription factor TFIIB.

A Drosophila cDNA encoding a human transcription factor TFIIB homologue was isolated by PCR methods. The deduced amino acid sequence indicates 85% sequence similarity with human TFIIB, and the corresponding cDNA product expressed in Escherichia coli is interchangeable with human TFIIB for both basal and GAL4-VP16-induced transcription. Structural motifs including the direct repeats, basic repeats, and sigma sequence similarities are well conserved among Drosophila, human, and Xenopus TFIIB. However, the N-terminal region of each direct repeat is less conserved among the three species, suggesting the presence of two structural subdomains in the direct repeat. Moreover, the amino acid changes in the N-terminal subdomain produce altered positions of the conserved amino acids between the direct repeats. An overall similarity in general structural features between TFIIB and TFIID tau (the TATA-binding subunit of TFIID) was previously noted. However, in contrast to the sequence divergence reported for the N-terminal domains of TFIID tau from different species, the N-terminal sequence of TFIIB was highly conserved among the species. This suggests that TFIIB has a more rigid structure, consistent with its function as a "bridging" protein between TFIID and RNA polymerase II. Further implications of the TFIIB structure are discussed.

Amino Acid Sequence↗
PubMed1992

A bipartite DNA binding domain composed of direct repeats in the TATA box binding factor TFIID.

Point mutations in residues comprising the interrupted direct repeats of TFIID eliminated DNA binding in an electrophoretic mobility shift assay. In contrast, mutations in nonconserved residues within the direct repeat regions or in lysine residues comprising the intervening basic repeat had no effect on DNA binding. However, small spacing changes (addition or deletion of one to three residues) in the basic repeat eliminated DNA binding. These results argue for a bipartite DNA binding domain composed of direct repeats with a strict spacing and orientation. Surprisingly, some direct repeat mutations that inhibited DNA binding failed to show a corresponding inhibition of basal transcription, indicating compensating interactions of TFIID with other general factors. The implications of these and other recent results for TFIID structure, promoter recognition, and interactions with other factors are discussed.

Amino Acid Sequence↗
PubMed1992

Transcription factor TFIID induces DNA bending upon binding to the TATA element.

The TATA box-binding factor TFIID plays a primary role in the process of transcription initiation by RNA polymerase II and its regulation by various gene-specific factors. Here we employ a permuted binding site/gel retardation assay with recombinant yeast and human TFIID to show that this factor induces DNA bending around the TATA element. These results are consistent with the presence of G + C-rich sequence elements flanking the consensus TATA element and led to the recently confirmed suggestion that TFIID interacts with the TATA element via the minor groove. They also raise the possibility that TFIID-induced bending might facilitate promoter interactions of other general factors in the preinitiation complex or interactions between general transcription factors and regulatory factors bound at upstream sites.

Base Sequence↗
PubMed1992

Identification of USF as the ubiquitous murine factor that binds to and stimulates transcription from the immunoglobulin lambda 2-chain promoter.

To study the specificity and identity of NF-lambda 2, a ubiquitous murine nuclear factor that interacts specifically with the promoter of the lambda 2-chain gene and stimulates its transcription, competition experiments were carried out using DNA fragments from various immunoglobulin regulatory elements. The results showed that a fragment containing the H-chain enhancer competed efficiently for the binding of NF-lambda 2. Dissection of the H-chain enhancer revealed that the microE3 motif contributed the competing ability. Additionally, a regulatory region found in the adenovirus major late promoter, which interacts with the human general transcription factor USF, competed very efficiently for binding to NF-lambda 2. This region contains a sequence, CACGTGAC, which is identical to a region within the NF-lambda 2 motif. The pattern of complexes formation using oligonucleotide probes corresponding to the NF-lambda 2 and USF motifs were identical, and they both differed from that displayed by the E3 probes. Antisera against different domains of USF also react specifically with NF-lambda 2 showing that this factor is antigenically related, if not identical, to USF. Furthermore, the activity of the lambda 2 promoter in an in vitro transcription assay was significantly reduced when the nuclear extract used was USF-depleted. Addition of exogenous USF to this extract restored the transcription activity. Therefore, we conclude that NF-lambda 2 is the murine homologue of USF.

Animals↗
PubMed1992

HIV-1 Tat acts as a processivity factor in vitro in conjunction with cellular elongation factors.

The HIV-1 trans-activator Tat increases the rate of transcription from the HIV-1 LTR promoter through the stem-loop-containing TAR RNA. To analyze the mechanisms of Tat action, a cell-free trans-activation system with no preincubation has been developed. Recombinant Tat specifically increased the level of a long runoff transcript but not a promoter-proximal transcript in a TAR-dependent fashion. These observations and the result of pulse-chase experiments support strongly the hypothesis that Tat enhances the ability of RNA polymerase to elongate over longer distances. Increased levels of the purified cellular factor TFIIF, essential for initiation and also implicated in elongation of transcription, obviated trans-activation by Tat by increasing the basal (Tat-independent) activity. However, another elongation factor, ATN/TFIIS, showed synergistic activation with Tat. An antiserum against a recombinant form of the large subunit of TFIIF (RAP 74) preferentially suppressed the activated level of transcription exerted by Tat. We propose the hypothesis that Tat acts as a processivity factor on RNA polymerase II in an analogous manner to TFIIF.

Amino Acid Sequence↗