Biomedical subjects

R G Roeder

Publications and source records attributed to R G Roeder.

At least 109 records · Page 6Linked to original sources

PubMed1996

The role of general initiation factors in transcription by RNA polymerase II.

Transcription initiation on protein-encoding genes represents a major control point for gene expression in eukaryotes, and is mediated by RNA polymerase II and a surprisingly complex array of general initiation factors (TFIIA, -B, -D, -E, -F and -H) that are highly conserved from yeast to man. Elucidation of structural and functional features of these factors on model promoters has revealed insights into biochemical mechanisms and provides a basis for understanding their regulation on diverse promoters by gene- and cell-specific activators.

Animals↗

PubMed1995

Core promoter-specific function of a mutant transcription factor TFIID defective in TATA-box binding.

In conjunction with other general initiation factors, the TATA box-binding protein (TBP) can direct basal transcription by RNA polymerase II from TATA-containing promoters, but its stable interaction with TBP-associated factors (TAFs) in the TFIID complex is required both for activator-dependent transcription and for basal transcription directed by an initiator element. We have generated a TATA-binding-defective TFIID complex containing an amino acid substitution in the DNA-binding surface of its TBP subunit. This mutated TFIID is defective in both basal and activated transcription from core promoters containing only a TATA box but supports transcription from initiator-containing promoters independently of the presence or absence of a TATA sequence. Our results show that a functional initiator element is needed to bypass the requirement for an active TATA DNA-binding surface in TFIID and imply that gene-specific transcription can be achieved by modulating distinct core promoter-specific TFIID functions--e.g., TBP-TATA versus TAF-initiator interactions.

Amino Acid Sequence↗

PubMed1995

Crystal structure of a TFIIB-TBP-TATA-element ternary complex.

The crystal structure of the transcription factor IIB (TFIIB)/TATA box-binding protein (TBP)/TATA-element ternary complex is described at 2.7 A resolution. Core TFIIB resembles cyclin A, and recognizes the preformed TBP-DNA complex through protein-protein and protein-DNA interactions. The amino-terminal domain of core TFIIB forms the downstream surface of the ternary complex, where it could fix the transcription start site. The remaining surfaces of TBP and the TFIIB can interact with TBP-associated factors, other class II initiation factors, and transcriptional activators and coactivators.

Adenoviridae↗

PubMed1995

Evolutionary conservation of human TATA-binding-polypeptide-associated factors TAFII31 and TAFII80 and interactions of TAFII80 with other TAFs and with general transcription factors.

Human transcription initiation factor TFIID is composed of the TATA-binding polypeptide (TBP) and at least 13 TBP-associated factors (TAFs) that collectively or individually are involved in activator-dependent transcription. To investigate protein-protein interactions involved in TFIID assembly and in TAF-mediated activator functions, we have cloned and expressed cDNAs encoding human TAFII80 and TAFII31. Coimmunoprecipitation assays showed that TAFII80 interacted with TAFII250, TAFII31, TAFII20, and TBP, but not with TAFII55. Similar assays showed that TAFII80 interacted with TFIIE alpha and with TFIIF alpha (RAP74) but not with TFIIB, TFIIE beta, or TFIIF beta (RAP30). Further studies with TAFII80 mutations revealed three distinct interaction domains which fall within regions conserved in human TAFII80, Drosophila TAFII60, and yeast TAFII60. The N terminus of TAFII80 (residues 1-100) interacts with both TAFII31 and TAFII20, while two C-terminal regions are involved, respectively, in interactions with TAFII250 and TFIIF alpha (RAP74) (residues 203-276) and with TBP and TFIIE alpha (residues 377-505). The interactions between TAFII80 and general factors TFIIE alpha and TFIIF alpha (RAP74) could be important for recruitment of GTFs during activator-dependent transcription. Because TAFs 80, 31, and 20 show sequence similarities to histones H4, H3, and H2B, as well as some parallel interactions, this subset of TAFs may form a related core structure within TFIID.

Amino Acid Sequence↗

PubMed1995

Structure and function of a human transcription factor TFIIIB subunit that is evolutionarily conserved and contains both TFIIB- and high-mobility-group protein 2-related domains.

Transcription factor TFIIIB plays a central role in transcription initiation by RNA polymerase III on genes encoding tRNA, 5S rRNA, and other small structural RNAs. We report the purification of a human TFIIIB-derived complex containing only the TATA-binding polypeptide (TBP) and a 90-kDa subunit (TFIIIB90) and the isolation of a cDNA clone encoding the 90-kDa subunit. The N-terminal half of TFIIIB90 exhibits sequence similarity to the yeast TFIIIB70 (BRF) and the class II transcription factor TFIIB and interacts weakly with TBP. The C-terminal half of TFIIIB90 contains a high-mobility-group protein 2 (HMG2)-related domain and interacts strongly with TBP. Recombinant TFIIIB90 plus recombinant human TBP substitute for human TFIIIB in a complementation assay for transcription of 5S, tRNA, and VA1 RNA genes, and both the TFIIB-related domain and the HMG2-related domain are required for this activity. TFIIIB90 is also required for transcription of human 7SK and U6 RNA genes by RNA polymerase III, but apparently within a complex distinct from the TBP/TFIIIB90 complex.

Amino Acid Sequence↗

PubMed1995

Control of transcription by Krüppel through interactions with TFIIB and TFIIE beta.

The zinc-finger protein Krüppel (Kr) is an integral part of the Drosophila segmentation gene cascade and is essential in organogenesis during later embryonic development. In tissue culture, Kr regulates transcription. Monomeric Kr can act as a transcriptional activator, whereas Kr dimers formed at high concentrations cause repression. Here we show that Kr-dependent control of transcription involves functional interactions with components of the basal RNA polymerase II transcription machinery, which includes the initiation factors TFIIA, B, E, F, H and I (refs 10, 11) as well as the TATA-binding protein (TBP) and TBP-associated factors (TAFs) contained in the multisubunit TFIID (ref. 12). Our results indicate that when acting from a site close to a basal promoter, monomeric Kr interacts with TFIIB to activate transcription, whereas an interaction of the Kr dimer with TFIIE beta, a subunit of TFIIE, results in transcriptional repression.

Animals↗

PubMed1995

TATA-binding protein residues implicated in a functional interplay between negative cofactor NC2 (Dr1) and general factors TFIIA and TFIIB.

The TATA-binding protein (TBP) plays a key role in transcription initiation. Several negative cofactors (NC1, NC2, and Dr1) are known to interact with TBP in a manner that prevents productive interactions of transcription factors TFIIA and TFIIB with promoter-bound TBP. To gain insights into the regulatory interplay on the surface of TBP, we have employed mutant forms of TBP to identify amino acid residues important for interactions with the negative regulatory cofactor NC2 and the general factor TFIIB. The results show the involvement of distinct domains of TBP in these interactions. Residues (Lys-133, Lys-145, and Lys-151) in the basic repeat region are important for interactions with NC2, as well as with TFIIA (Buratowski, S., and Zhou, H. (1992) Science 255, 1130-1132; Lee, D. K., DeJong, J., Hashimoto, S., Horikoshi, M., and Roeder, R. G. (1992) Mol. Cell. Biol. 12, 5189-5196), whereas a residue (Leu-189) in the second stirrup-like loop spanning S2' and S3' is required for interaction with TFIIB. In addition, we demonstrate that NC2 is identical to the previously cloned negative cofactor Dr1. The implications of these results for TBP structure and function are discussed.

Binding Sites↗

PubMed1995

Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription.

The human general transcription factor TFIIA is one of several factors involved in specific transcription by RNA polymerase II, possibly by regulating the activity of the TATA-binding subunit (TBP) of TFIID. TFIIA purified from HeLa extracts consists of 35-, 19-, and 12-kDa subunits. Here we describe the isolation of a cDNA clone (hTFIIA gamma) encoding the 12-kDa subunit. Using expression constructs derived from hTFIIA gamma and TFIIA alpha/beta (which encodes a 55-kDa precursor to the alpha and beta subunits of natural TFIIA), we have constructed a synthetic TFIIA with a polypeptide composition similar to that of natural TFIIA. The recombinant complex supports the formation of a DNA-TBP-TFIIA complex and mediates both basal and Gal4-VP16-activated transcription by RNA polymerase II in TFIIA-depleted nuclear extracts. In contrast, TFIIA has no effect on tRNA and 5S RNA transcription by RNA polymerase III in this system. We also present evidence that both the p55 and p12 recombinant subunits interact with TBP and that the basic region of TBP is critical for the TFIIA-dependent function of TBP in nuclear extracts.

Amino Acid Sequence↗

PubMed1995

Cloning and characterization of a TFIIIC2 subunit (TFIIIC beta) whose presence correlates with activation of RNA polymerase III-mediated transcription by adenovirus E1A expression and serum factors.

TFIIIC2 is a general factor essential for transcription of 5S RNA, tRNA, and VA RNA genes by mammalian RNA polymerase III and consists of two forms designated TFIIIC2a and TFIIIC2b. TFIIIC2a and TFIIIC2b share common subunits of 220, 102, 90, and 63 kD but differ with respect to transcription activity and the presence of a presumptive 110-kD subunit in the active form (TFIIIC2a). Because both forms can bind the promoter directly, a selective role for the 110-kD subunit in the regulation of RNA polymerase III activity has been suggested. To investigate this possibility, we have cloned and expressed a cDNA encoding the 110-kD subunit (TFIIIC beta). Immunoprecipitation studies with anti-TFIIIC beta antibodies have confirmed that TFIIIC beta is a bona fide subunit present only in TFIIIC2a, that TFIIIC2a and the general factor TFIIIC1 are associated in unfractionated extracts, and that previously undetected polypeptides (potential TFIIIC1 subunits) can be isolated in association with TFIIIC2a. Previous studies have shown that increases in RNA polymerase III activity during infection of cells by adenovirus (with concomitant E1A expression) or during cell growth at high serum concentration results from an increased activity in the TFIIIC fraction. Studies with antibodies to TFIIIC beta have shown that this is strongly correlated with a selective increase in the cellular concentration of the TFIIIC beta 110-kD subunit and a concomitant rise in the ratio of the active-to-inactive forms of TFIIIC2.

Adenoviridae↗

PubMed1995

A novel LBP-1-mediated restriction of HIV-1 transcription at the level of elongation in vitro.

The cellular factor, LBP-1, can repress HIV-1 transcription by preventing the binding of TFIID to the promoter. Here we have analyzed the effect of recombinant LBP-1 on HIV-1 transcription in vitro by using a "pulse-chase" assay. LBP-1 had no effect on initiation from a preformed preinitiation complex and elongation to position +13 ("pulse"). However, addition of LBP-1 after RNA polymerase was stalled at +13 strongly inhibited further elongation ("chase") by reducing RNA polymerase processivity. Severe mutations of the high affinity LBP-1 binding sites between -4 and +21 did not relieve the LBP-1-dependent block. However, LBP-1 could bind independently to upstream low affinity sites (-80 to -4), suggesting that these sites mediate the effect of LBP-1 on elongation. These results demonstrate a novel function of LBP-1, restricting HIV-1 transcription at the level of elongation. In addition, Tat was found to suppress the antiprocessivity effect of LBP-1 on HIV-1 transcription in nuclear extracts. These findings strongly suggest that LBP-1 may provide a natural mechanism for restricting the elongation of HIV-1 transcripts and that this may be a target for the action of Tat in enhancing transcription.

Base Sequence↗

PubMed1995

Cloning of an intrinsic human TFIID subunit that interacts with multiple transcriptional activators.

TFIID is a multisubunit protein complex comprised of the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs). The TAFs in TFIID are essential for activator-dependent transcription. The cloning of a complementary DNA encoding a human TFIID TAF, TAFII55, that has no known homolog in Drosophila TFIID is now described. TAFII55 is shown to interact with the largest subunit (TAFII230) of human TFIID through its central region and with multiple activators--including Sp1, YY1, USF, CTF, adenoviral E1A, and human immunodeficiency virus-type 1 Tat proteins--through a distinct amino-terminal domain. The TAFII55-interacting region of Sp1 was localized to its DNA-binding domain, which is distinct from the glutamine-rich activation domains previously shown to interact with Drosophila TAFII110. Thus, this human TFIID TAF may be a co-activator that mediates a response to multiple activators through a distinct mechanism.

Adenovirus E1A Proteins↗

PubMed1995

The upstream stimulatory factor functionally interacts with the Alzheimer amyloid beta-protein precursor gene.

The amyloid beta-protein precursor (APP) gives rise to the A beta peptide, which is deposited in the brains of patients with Alzheimer's disease and Down's syndrome. Overexpression of APP due to a third copy of the gene appears to correlate with very early onset of Alzheimer's disease neuropathology in the brains of Down's syndrome patients. Thus, the identification of the factors involved with transcriptional regulation of the APP gene could provide critical clues regarding the events leading to the formation of amyloid deposits. An overlapping AP-1/AP-4 site in the proximal promoter region (-39 to -49) of the human APP gene has previously been shown to increase transcription 4-fold. Here we identify the factor binding specifically to this element as the upstream stimulatory factor USF, unrelated to the c-fos/c-jun complex or the AP-4 factor. In vitro transcription and co-transfection studies show that USF activates transcription from the APP promoter and that the AP-1/AP-4 element participates in this activation. Modulation of APP expression via regulation of USF could potentially ameliorate the production of Alzheimer-augmented beta-amyloid.

Alzheimer Disease↗

PubMed1995

Proximal sequence element-binding transcription factor (PTF) is a multisubunit complex required for transcription of both RNA polymerase II- and RNA polymerase III-dependent small nuclear RNA genes.

The proximal sequence element (PSE), found in both RNA polymerase II (Pol II)- and RNA Pol III-transcribed small nuclear RNA (snRNA) genes, is specifically bound by the PSE-binding transcription factor (PTF). We have purified PTF to near homogeneity from HeLa cell extracts by using a combination of conventional and affinity chromatographic methods. Purified PTF is composed of four polypeptides with apparent molecular masses of 180, 55, 45, and 44 kDa. A combination of preparative electrophoretic mobility shift and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses has conclusively identified these four polypeptides as subunits of human PTF, while UV cross-linking experiments demonstrate that the largest subunit of PTF is in close contact with the PSE. The purified PTF activates transcription from promoters of both Pol II- and Pol III-transcribed snRNA genes in a PSE-dependent manner. In addition, we have investigated factor requirements in transcription of Pol III-dependent snRNA genes. We show that in extracts that have been depleted of TATA-binding protein (TBP) and associated factors, recombinant TBP restores transcription from U6 and 7SK promoters but not from the VAI promoter, whereas the highly purified TBP-TBP-associated factor complex TFIIIB restores transcription from the VAI but not the U6 or 7SK promoter. Furthermore, by complementation of heat-treated extracts lacking TFIIIC activity, we show that TFIIIC1 is required for transcription of both the 7SK and VAI genes, whereas TFIIIC2 is required only for transcription of the VAI gene. From these observations, we conclude (i) that PTF and TFIIIC2 function as gene-specific as gene-specific factors for PSE-and B-box-containing Pol III genes, respectively, (ii) that the form of TBP used by class III genes with upstream promoter elements differs from the from used by class III genes with internal promoters, and (iii) that TFIIIC1 is required for both internal and external Pol III promoters.

Base Sequence↗

PubMed1995

Cloning, functional characterization, and mechanism of action of the B-cell-specific transcriptional coactivator OCA-B.

Biochemical purification and cognate cDNA cloning studies have revealed that the previously described transcriptional coactivator OCA-B consists of a 34- or 35-kDa polypeptide with sequence relationships to known coactivators that function by protein-protein interactions. Studies with a recombinant protein have proved that a single OCA-B polypeptide is the main determinant for B-cell-specific activation of immunoglobulin (Ig) promoters and provided additional insights into its mechanism of action. Recombinant OCA-B can function equally well with Oct-1 or Oct-2 on an Ig promoter, but while corresponding POU domains are sufficient for OCA-B interaction, and for octamer-mediated transcription of a histone H2B promoter, an additional Oct-1 or Oct-2 activation domain(s) is necessary for functional synergy with OCA-B. Further studies additional Oct-1 or Oct-2 activation domain(s) is necessary for functional synergy with OCA-B. Further studies show that Ig promoter activation by Oct-1 and OCA-B requires still other general (USA-derived) cofactors and also provide indirect evidence that distinct Oct-interacting cofactors regulate H2B transcription.

Amino Acid Sequence↗

PubMed1995

Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure-function studies of TFIIE-alpha.

The general transcription factor TFIIE recruits TFIIH at a late stage of transcription initiation complex formation and markedly stimulates TFIIH-dependent phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II. To study this function of TFIIE in more detail, systematic deletion mutations were introduced into the large subunit of TFIIE (TFIIE-alpha) and were analyzed with regard to their effects on TFIIH-dependent CTD phosphorylation, TFIIE-dependent basal and enhancer-dependent transcription, and interactions of TFIIE-alpha with both TFIIE-beta and TFIIH. The amino (N)-terminal half of TFIIE-alpha, which possesses several putative structural motifs, was sufficient for the phosphorylation and transcription activities and for TFIIE-beta interactions, whereas a site effecting both strong interactions with TFIIH and large stimulatory effects on transcription and CTD phosphorylation was localized to an acidic region near the carboxy (C) terminus. The fact that these activities appear to be tightly linked supports the idea that TFIIE interacts physically and functionally with TFIIH and that CTD phosphorylation is essential for transcription under normal conditions. The present results suggest that TFIIE, via its effect on TFIIH, may act as a checkpoint for formation of a preinitiation complex.

Base Sequence↗

PubMed1994

Phosphorylation negatively regulates the function of coactivator PC4.

Human positive cofactor 4 (PC4) mediates activator-dependent transcription by RNA polymerase II, apparently through interactions with transcriptional activators and the basal transcription machinery. We report here that PC4 function is modulated by in vivo phosphorylation. Protein-protein interaction studies and in vitro transcription assays demonstrate that only the nonphosphorylated form of PC4 is functionally active. Although recombinant PC4 can be phosphorylated by casein kinase II and protein kinase C in vitro, mutational and mass spectrometric analyses suggest that the in vivo hyperphosphorylation of PC4 is mediated mainly by casein kinase II and restricted to an N-terminal serine-rich region. These observations provide one example of a transcriptional cofactor that is negatively regulated by casein kinase II phosphorylation.

Amino Acid Sequence↗

PubMed1994

Transformation by homeobox genes can be mediated by selective transcriptional repression.

Altered transcription is a recurrent theme in the field of cancer biology. But despite the central role of transcription in transformation, little is known about the mechanism by which dominant nuclear oncogenes induce malignancies. Homeobox family proteins are prominent examples of transcriptional regulators which control development and can function as oncogenes. Here we explore the molecular basis for transformation by this class of regulators using Oct-2 and Oct-1. We show that the DNA binding POU domains of these proteins are selective and sequence-specific transcriptional repressors that produce malignant lymphomas when they are expressed in T cells of transgenic mice. Mutagenesis experiments identified a specific set of promoters, those containing octamer regulatory elements, as the targets for transformation by selective inhibition of gene expression.

Animals↗

PubMed1994

Overlapping transcription by RNA polymerases II and III of the Xenopus TFIIIA gene in somatic cells.

Xenopus TFIIIA gene expression is developmentally regulated. The per cell level of steady-state TFIIIA mRNA present in oocytes is drastically reduced (10(6)-fold) in adult somatic cells, suggesting regulation at the transcriptional level. This is supported by the presence of TFIIIA mRNAs with different 5'-ends in oocytes and in somatic cells, which suggests the presence of distinct cell-/stage-specific promoters, i.e. a strong oocyte-specific and a weak somatic-specific promoter. Here, by mapping the 5'-ends of TFIIIA RNAs found in Xenopus somatic cells, we document the activity not only of an upstream somatic-specific promoter, but also of a down-stream "oocyte-specific" promoter that gives rise to a minor population of somatic TFIIIA mRNAs initiated from the same transcription start site utilized in oocytes. This result explains the presence of an oocyte-type form of TFIIIA protein in somatic cells. Furthermore, by characterizing transcription from the Xenopus TFIIIA promoter in somatic cell nuclear extracts and comparing the in vitro transcription start sites with the 5'-ends of endogenous somatic TFIIIA transcripts in poly(A)+ and poly(A)- RNA fractions, we show that RNA polymerase III initiates at position -70 of the "oocyte-specific" core promoter and transcribes through the promoter and into the TFIIIA coding sequences in somatic cells. These results suggest a possible polymerase III transcription-mediated down-regulation of the oocyte-specific TFIIIA promoter in Xenopus somatic cells.

Animals↗