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PubMed2005

The growing pre-mRNA recruits actin and chromatin-modifying factors to transcriptionally active genes.

In the dipteran Chironomus tentans, actin binds to hrp65, a nuclear protein associated with mRNP complexes. Disruption of the actin-hrp65 interaction in vivo by the competing peptide 65-2CTS reduces transcription drastically, which suggests that the actin-hrp65 interaction is required for transcription. We show that the inhibitory effect of the 65-2CTS peptide on transcription is counteracted by trichostatin A, a drug that inhibits histone deacetylation. We also show that actin and hrp65 are associated in vivo with p2D10, an evolutionarily conserved protein with histone acetyltransferase activity that acts on histone H3. p2D10 is recruited to class II genes in a transcription-dependent manner. We show, using the Balbiani ring genes of C. tentans as a model system, that p2D10 is cotranscriptionally associated with the growing pre-mRNA. We also show that experimental disruption of the actin-hrp65 interaction by the 65-2CTS peptide in vivo results in the release of p2D10 from the transcribed genes, reduced histone H3 acetylation, and a lower level of transcription activity. Furthermore, antibodies against p2D10 inhibit run-on elongation. Our results suggest that actin, hrp65, and p2D10 are parts of a positive feedback mechanism that contributes to maintaining the active transcription state of a gene by recruiting HATs at the RNA level.

Acetyltransferases↗
PubMed1990

Transcription of a yeast U6 snRNA gene requires a polymerase III promoter element in a novel position.

Vertebrate genes coding for U6 small nuclear RNA are transcribed by RNA polymerase III (pol III), using only upstream promoter elements rather than the A and B block internal control regions typical of most pol III transcription units. We show that expression of the U6 gene from the yeast Saccharomyces cerevisiae has two unexpected features: it requires a B block promoter element, and this element is located in a novel position, 120 bp downstream of the coding region. In tRNA genes, the B block is the primary binding site for transcription factor (TF) IIIC, whose function is to promote the subsequent binding of TFIIIB. Both factors are thus implicated in yeast U6 gene transcription. We present a model of the U6 transcription complex based on the structure of yeast and vertebrate U6 promoters.

Base Sequence↗
PubMed1988

Developmental regulation of two 5S ribosomal RNA genes.

The developmental regulation of two kinds of Xenopus 5S RNA genes (oocyte and somatic types) can be explained by differences in the stability of protein-protein and protein-DNA interactions in a transcription complex that directs transcription initiation by RNA polymerase III. Dissociation of transcription factors from oocyte 5S RNA genes during development allows them to be repressed by chromatin assembly. In the same cells, somatic 5S RNA genes remain active because their transcription complexes are stable.

Animals↗
PubMed1994

Differential regulation of RNA polymerases I, II, and III by the TBP-binding repressor Dr1.

RNA polymerases I, II, and III each use the TATA-binding protein (TBP). Regulators that target this shared factor may therefore provide a means to coordinate the activities of the three nuclear RNA polymerases. The repressor Dr1 binds to TBP and blocks the interaction of TBP with polymerase II- and polymerase III-specific factors. This enables Dr1 to coordinately regulate transcription by RNA polymerases II and III. Under the same conditions, Dr1 does not inhibit polymerase I transcription. By selectively repressing polymerases II and III, Dr1 may shift the physiological balance of transcriptional output in favor of polymerase I.

Base Sequence↗
PubMed1991

Transcription complex disruption caused by a transition in chromatin structure.

Chromatin structure is known to influence class III gene expression in vitro. We describe the active transcription of Xenopus class III genes following replication and assembly into chromatin by using Xenopus egg extracts. Changes in the structure of this active chromatin dependent on the presence of exogeneous Mg2+ ATP or on the addition of a mixture of histones H2A and H2B are shown to lead to the selective repression of Xenopus 5S RNA genes. Preexisting transcription complexes on 5S DNA are disrupted following the reorganization of a "disordered" histone-DNA complex into a structure consisting of physiologically spaced nucleosomes. Thus, we demonstrate that chromatin structural transitions can have dominant and specific effects on transcription.

Adenosine Triphosphate↗
PubMed1993

Orientation and topography of RNA polymerase III in transcription complexes.

A photo-cross-linking method has been used to map the subunits of Saccharomyces cerevisiae RNA polymerase (Pol) III with respect to DNA in binary (preinitiation) and ternary (RNA-elongating) transcription complexes. Transcription factor- and Pol III-containing complexes have been assembled on S. cerevisiae SUP4 tRNA(Tyr) gene probes containing the photoactive nucleotide 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP in different specified positions. Covalent DNA-protein linkages form upon irradiation of these complexes, and the Pol III subunits that are cross-linked to individual positions in the SUP4 tRNA gene have been identified. RNA Pol III cross-linking has been shown to require the box B downstream promoter element of the tRNA gene and the presence of transcription factor TFIIIB. Further proof of specificity has been provided by demonstrating that particular Pol III subunits move out of the range of upstream-placed photoactive nucleotides, and that others move into the range of downstream-placed photoactive nucleotides, as a consequence of initiating and elongating RNA chains. Binding and specific placement of Pol III have also been shown to require both the B' and the B" components of TFIIIB. Nine Pol III subunits are cross-linked from different positions of the SUP4 tRNA gene's nontranscribed strand. In binary transcription complexes, the two largest Pol III subunits are accessible to photo-cross-linking over the entire stretch of the DNase I footprint. The 27- and 34-kDa Pol III subunits are also relatively extended along DNA; its upstream projection makes the 34-kDa subunit a candidate for interaction with TFIIIB, while the 27-kDa subunit is accessible to photo-cross-linking from the leading edge of the Pol III binding site. Several subunits, including the 82- and 53-kDa subunits in binary transcription complexes, are relatively localized in their accessibility to cross-linking. Multiple Pol III subunits are accessible to specific cross-linking from a single photoactive nucleotide in the middle of the transcription bubble of an arrested ternary transcription complex. It is suggested that this precisely placed transcription complex comprises a dynamic ensemble of structural states rather than a single perfectly constrained entity.

Base Sequence↗
PubMed1994

Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

Animals↗
PubMed1995

The activity of transcription factor PBP, which binds to the proximal sequence element of mammalian U6 genes, is regulated during differentiation of F9 cells.

Mouse F9 embryonic carcinoma (EC) cells differentiate in culture to parietal endoderm (PE) cells upon induction with retinoic acid and cyclic AMP. In the course of this process, the expression of polymerase III transcripts, e.g., 5S rRNA and U6 small nuclear RNA, is dramatically reduced. This reduction of endogenous RNA content is accompanied by a loss of transcriptional capacity in cell extracts from PE cells. Partial purification of such extracts reveals that the DNA-binding activity of transcription factor PBP, binding specifically to the proximal sequence element (PSE) sequence of vertebrate U6 genes, is significantly reduced. This finding is corroborated by a loss in the transcriptional activity of this factor in reconstitution assays with partially purified polymerase III transcription components. In contrast, the activity of TFIIIA and TFIIIB and the amount of free TATA-binding protein remain unchanged during the differentiation process analyzed here. These data show for the first time that the PSE-binding protein PBP is essentially involved in the differential regulation of polymerase III genes governed by external promoters.

Animals↗
PubMed1996

TFIIIC1 acts through a downstream region to stabilize TFIIIC2 binding to RNA polymerase III promoters.

An in vitro system reconstituted with highly purified RNA polymerase III, TFIIIC2, and TFIIIB has been used to identify two chromatographically distinct human RNA polymerase III transcription factors, TFIIIC1 and TFIIIC1', which are functionally equivalent to the previously defined TFIIIC1 (S. T. Yoshinaga, P. A. Boulanger, and A. J. Berk, Proc. Natl. Acad. Sci. USA 84:3585-3589, 1987). Interactions between TFIIIC2, TFIIIC1 (or TFIIIC1'), and the VA1 and tRNA1(Met) templates have been investigated by DNase I footprint analysis. Homogeneous TFIIIC2 alone shows only a weak footprint over the B-box region of the VA1 and tRNA1(Met) templates, whereas TFIIIC1 (or TFIIIC1') alone shows both a strong interaction over the downstream termination region and a very weak interaction near the A-box region. Importantly, when both factors are present simultaneously, TFIIIC1 (or TFIIIC1') dramatically enhances the level of TFIIIC2 binding and extends the footprint to a region that includes the A box. The downstream termination region is essential for this cooperative interaction between TFIIIC2 and TFIIIC1 (or TFIIIC1') on the VA1 and tRNA1(Met) templates and plays a role in the overall accuracy and efficiency of RNA polymerase III transcription.

Binding Sites↗
PubMed1999

Activation of RNA polymerase III transcription in cells transformed by simian virus 40.

RNA polymerase (Pol) III transcription is abnormally active in fibroblasts that have been transformed by simian virus 40 (SV40). This report presents evidence that two separate components of the general Pol III transcription apparatus, TFIIIB and TFIIIC2, are deregulated following SV40 transformation. TFIIIC2 subunits are expressed at abnormally high levels in SV40-transformed cells, an effect which is observed at both protein and mRNA levels. In untransformed fibroblasts, TFIIIB is subject to repression through association with the retinoblastoma protein RB. The interaction between RB and TFIIIB is compromised following SV40 transformation. Furthermore, the large T antigen of SV40 is shown to relieve repression by RB. The E7 oncoprotein of human papillomavirus can also activate Pol III transcription, an effect that is dependent on its ability to bind to RB. The data provide evidence that both TFIIIB and TFIIIC2 are targets for activation by DNA tumor viruses.

3T3 Cells↗
PubMed2000

Retinoblastoma protein disrupts interactions required for RNA polymerase III transcription.

The retinoblastoma protein (RB) has been shown to suppress RNA polymerase (Pol) III transcription in vivo (R. J. White, D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides, Nature 382:88-90, 1996). This regulation involves interaction with TFIIIB, a multisubunit factor that is required for the expression of all Pol III templates (C. G. C. Larminie, C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P. Jackson, and R. J. White, EMBO J. 16:2061-2071, 1997; W.-M. Chu, Z. Wang, R. G. Roeder, and C. W. Schmid, J. Biol. Chem. 272:14755-14761, 1997). However, it has not been established why RB binding to TFIIIB results in transcriptional repression. For several Pol II-transcribed genes, RB has been shown to inhibit expression by recruiting histone deacetylases, which are thought to decrease promoter accessibility. We present evidence that histone deacetylases exert a negative effect on Pol III activity in vivo. However, RB remains able to regulate Pol III transcription in the presence of the histone deacetylase inhibitor trichostatin A. Instead, RB represses by disrupting interactions between TFIIIB and other components of the basal Pol III transcription apparatus. Recruitment of TFIIIB to most class III genes requires its binding to TFIIIC2, but this can be blocked by RB. In addition, RB disrupts the interaction between TFIIIB and Pol III that is essential for transcription. The ability of RB to inhibit these key interactions can explain its action as a potent repressor of class III gene expression.

Animals↗
PubMed1989

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↗
PubMed2001

Comparative mapping of five coding DNA sequences on cattle chromosomes 7 and 25.

Comparative mapping of four genes and one unknown coding DNA sequence in breakpoint positions of bovine chromosomes (BTA) 7 and 25 are presented. Performing a genome data base search five bovine expressed sequence tags from the MARC library matched with human genes coding for the general transcription factor IIIC polypeptide 1 (GTF3C1), the hypothetical protein KIAA0556, the interleukin 4 receptor (IL4R), the regulatory factor X-associated ankyrin-containing protein (RFXANK), and with an unknown human coding sequence partially homologous to the genomic cosmid clone R30923. Loci for these sequences were COMPASS predicted on BTA7 or BTA18 and to BTA18 or BTA25. Mapping was performed in a cattle-hamster somatic hybrid cell panel and a cattle-hamster 5000 rad whole genome radiation hybrid panel. GTF3C1, KIAA0556 and IL4R were assigned to the centromere region of BTA25 and RFXANK and R30923 close to the centromere of BTA7. The assignments contribute to the identification of evolutionary chromosome break points between human chromosomes 16 and 19 and BTA7, BTA18, and BTA25.

Animals↗
PubMed1991

Analysis of the molecular mechanisms for the species-specific transcription of Drosophila and human tRNA gene transcription components.

The transcription of eucaryotic tRNA genes requires two factors IIIB and IIIC, in addition to RNA polymerase III, to reconstitute this process in vitro. We have examined the functional exchangeability of these components from Drosophila and human systems. The reconstitution of heterologous IIIB and IIIC components demonstrated that neither factor will functionally substitute for the homologous components to activate tRNA gene transcription. The addition of the heterologous Drosophila factors to HeLa transcription assays causes an inhibition of RNA synthesis that is dependent upon the order of addition of these proteins to the DNA template. Thus, it appears that tRNA gene transcription in these systems is species-specific. We have further analyzed the reason for the apparent incompatibilities of these components by the use of stable complex formation assays. We find that human HeLa IIIB and Drosophila IIIC are unable to form stably associated complexes with a tRNA gene template, whereas the Drosophila IIIB and HeLa IIIC do form stable but nonproductive complexes. These results demonstrate that specific IIIC-IIIB interactions are critical in the formation of productive transcription complexes and are responsible for the observed species specificity of Drosophila and human tRNA gene transcription.

Animals↗
PubMed1990

The transcription complex of the Xenopus somatic 5 S RNA gene. A functional analysis of protein-DNA interactions outside of the internal control region.

We have defined protein-DNA interactions associated with the transcription complex of a Xenopus somatic 5 S RNA gene under efficient in vitro transcription conditions. Transcription factor IIIA, the internal control region of the 5 S RNA gene and specific DNA sequences 5' and 3' of the internal control region are all involved in the formation of a multiprotein complex. Specific protein-DNA interactions outside of the 5 S RNA gene itself, revealed by DNase I footprinting, have no apparent role in the transcription process. Sequences within the 5 S RNA gene, 5' of the internal control region, are not essential for transcription by RNA polymerase III in vitro, but do contribute to its efficiency.

Animals↗
PubMed1987

Differential transcription of Xenopus oocyte and somatic-type 5 S genes in a Xenopus oocyte extract.

We have measured the transcription of Xenopus laevis oocyte and somatic-type 5 S RNA genes in S-150 extracts prepared from ovaries of mature X. laevis females (Glikin, G. C., Ruberti, I., and Worcel, A. (1984) Cell 37, 33-41). We find that somatic-type 5 S genes are transcribed at least 40-fold more efficiently than oocyte-type 5 S genes. Since adenovirus VA, Xenopus OAX, and Xenopus tRNAMet genes are all transcribed at levels similar to that of the somatic-type 5 S gene, this difference reflects a low level of oocyte-type 5 S gene transcription. Somatic-type 5 S transcription is a linear function of somatic-type 5 S DNA concentration and this, together with the efficient transcription of other class III genes, suggests that RNA polymerase III and the general class III transcription factors are not limiting under the conditions used here. Moreover, the 5 S gene-specific transcription factor TFIIIA does not limit transcription under these conditions as preincubation with purified TFIIIA does not alter transcription of either gene, and both genes exhibit characteristic TFIIIA footprints under transcription conditions in the S-150. Somatic-type 5 S DNA incubated in the S-150 and then injected into oocyte nuclei is actively transcribed whereas oocyte-type 5 S DNA treated in the same manner is inactive. We conclude that factors in the S-150 distinguish between somatic and oocyte-type 5 S genes, assembling active complexes preferentially on the former and inactive complexes preferentially on the latter. This process is not explained by binding properties of transcription factors for these genes as currently understood and represents a previously unrecognized mechanism of transcriptional regulation of the Xenopus 5 S genes.

Animals↗