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PubMed1987

Multiple proteins bind to VA RNA genes of adenovirus type 2.

Using fractionated HeLa cell nuclear extracts and both nuclease (DNase I) cleavage and chemical cleavage (methidiumpropyl-EDTA X Fe(II) protection methodologies, we demonstrated the presence of three proteins which interacted specifically, yet differentially, with the two VA genes of adenovirus type 2. One, previously identified as transcription initiation factor TFIIIC, bound to a site centered on the transcriptionally essential B-block concensus element of the VAI gene and, with a lower affinity, to the analogous site in the VAII gene. Another, identified as the cellular protein involved in adenovirus replication, nuclear factor I, bound to sites immediately downstream from the two VAI terminators (at approximately +160 and +200). The third, a previously unrecognized VA gene binding protein termed VBP, bound immediately upstream of the B-block element in the VAI gene but showed no binding to VAII. Possible roles for these proteins in VA gene transcription were investigated in in vitro assay systems reconstituted with partially purified transcription factors (RNA polymerase III, TFIIIB, and TFIIIC). Although TFIIIC activity was present predominantly in fractions containing B-block binding activity, there was not complete correspondence between functional and DNA binding activities. The nuclear factor I-like protein had no effect when added to a complete transcription reaction. The presence of VBP appeared to depress the intrinsic ratio of VAI-VAII synthesis, thereby simulating the relative transcription levels observed early in adenovirus infection of HeLa cells. These observations suggest a model, involving both intragenic binding factors (VBP and TFIIIC) and variable template concentrations, for the differential regulation of VA transcription during the course of adenovirus infection.

Adenoviridae↗
PubMed1996

A suppressor of mutations in the class III transcription system encodes a component of yeast TFIIIB.

Class III genes depend on TFIIIB for recruitment of RNA polymerase III. Yeast TFIIIB is comprised of three components: TBP, TFIIIB70 and a 90 kDa polypeptide contained in the fraction B". We report the isolation of the yeast gene TFC7 which, based on genetic and biochemical evidence, encodes the 90 kDa polypeptide. TFC7 was isolated as a multicopy suppressor of temperature-sensitive mutations in the two largest subunits of TFIIIC. It is an essential gene, encoding a polypeptide of 68 kDa migrating with an apparent size of approximately 90 kDa. In gel shift assays, recombinant TFC7 protein (rTFC7) alone did not bind detectably to DNA, or to the TFIIIC-DNA complex even in the presence of TBP or TFIIIB70, but it was required to assemble the TFIIIB-TFIIIC-DNA complex. The two-hybrid assay pointed to an interaction between TFC7 protein and tau 131, the second largest subunit of TFIIIC (that also interacts with TFIIIB70). rTFC7p can replace the B" component of TFIIIB for synthesis of U6 RNA in a system reconstituted with recombinant TBP and TFIIIB70 polypeptides and highly purified RNA polymerase III. Surprisingly, specific transcription of the SUP4 tRNATyr gene promoted by rTFC7p was much weaker than with B". An additional factor activity, provided by the recently identified TFIIIE fraction, was required to restore control levels of transcription.

Amino Acid Sequence↗
PubMed1993

Basal promoter and enhancer element of yeast U6 snRNA gene.

The yeast U6 snRNA gene, SNR6, transcribed by RNA polymerase III or C, is shown to have a mixed promoter with upstream, intragenic and downstream elements. The distant downstream B block behaves as a typical enhancer element. Required in vivo, and for transcription of chromatin templates in vitro, it was also active in reversed orientation. As shown by footprinting and electron microscopy, the factor TFIIIC, or tau, bound the B block in an oriented manner and was able to induce DNA looping. The factor TFIIIC appeared to act via a weak A block located at position +21. This A block-related motif was essential in vivo and with chromatin templates. When changed into a consensus A block it favored DNA looping by TFIIIC firmly anchored on the B block, and activated a B block lacking gene in vivo and in vitro. The role of the TATA box at -30 was most apparent using a purified transcription system. With the A block, it appeared to contribute to start site selection. The results suggest a model where three weak promoter elements collaborate to assemble the transcription complex by DNA looping and synergistic protein-DNA interactions.

Base Sequence↗
PubMed2001

Nhp6, an HMG1 protein, functions in SNR6 transcription by RNA polymerase III in S. cerevisiae.

Nhp6A and Nhp6B are HMG1-like proteins required for the growth of S. cerevisiae at elevated temperatures. We show that the conditional lethality of an nhp6 strain results from defective transcription of SNR6 (U6 snRNA) by RNA polymerase III. Overexpression of U6 snRNA or Brf1, a limiting component of TFIIIB, and an activating mutation (PCF1-1) in TFIIIC were each found to suppress the nhp6 growth defect. Additionally, U6 snRNA levels, which are reduced over 10-fold in nhp6 cells at 37 degrees C, were restored by Brf1 overexpression and by PCF1-1. Nhp6A protein specifically enhanced TFIIIC-dependent, but not TATA box-dependent, SNR6 transcription in vitro by facilitating TFIIIC binding to the SNR6 promoter. Thus, Nhp6 has a direct role in transcription complex assembly at SNR6.

DNA, Fungal↗
PubMed2003

The Brf1 and Bdp1 subunits of transcription factor TFIIIB bind to overlapping sites in the tetratricopeptide repeats of Tfc4.

The RNA polymerase III initiation factor TFIIIB is assembled onto DNA through interactions involving the Tfc4 subunit of the assembly factor TFIIIC and two subunits of TFIIIB, Brf1 and Bdp1. Tfc4 contains two arrays of tetratricopeptide repeats (TPRs), each of which provides a binding site for Brf1. Dominant mutations in the ligand binding channel of the first TPR array, TPRs1-5, and on the back side of this array, increase Brf1 binding by Tfc4. Here we examine the biological importance of the second TPR array, TPRs6 -9. Radical mutations at phylogenetically conserved residues in the ligand binding channel of TPRs6 -9 impair pol III reporter gene transcription. Biochemical studies on one such mutation, L469K in TPR7, revealed a defect in the recruitment of Brf1 into TFIIIB-TFIIIC-DNA complexes and diminished the direct interaction between Tfc4 and Brf1. Multicopy suppression analysis implicates TPR9 in Brf1 binding and TPRs7 and 8 in binding to more than one ligand. Indeed, the L469K mutation also decreased the binding affinity for Bdp1 incorporation into TFIIIB-TFIIIC-DNA complexes and inhibited binary interactions between Bdp1 and Tfc4. The Bdp1 binding domain in Tfc4 was mapped to TPRs1-9, a domain that contains both TPR arrays and thus overlaps two of the known binding sites for Brf1. The properties of the L469K mutation identify both Brf1 and Bdp1 as ligands for the second TPR array.

Binding Sites↗
PubMed2006

Epstein-Barr virus induces cellular transcription factors to allow active expression of EBER genes by RNA polymerase III.

The EBER genes of Epstein-Barr virus (EBV) are transcribed by RNA polymerase (pol) III to produce untranslated RNAs that are implicated in oncogenesis. These EBER transcripts are the most highly expressed viral gene products in EBV-transformed cells. We have identified changes to the cellular transcription machinery that may contribute to the high levels of EBER RNA. These include phosphorylation of ATF2, which interacts with EBER promoters. A second is induction of TFIIIC, a pol III-specific factor that activates EBER genes; all five subunits of TFIIIC are overexpressed in EBV-positive cells. In addition, EBV induces BDP1, a subunit of the pol III-specific factor TFIIIB. Although BDP1 is the only TFIIIB subunit induced by EBV, its induction is sufficient to stimulate EBER expression in vivo, implying a limiting function. The elevated levels of BDP1 and TFIIIC in EBV-positive cells stimulate production of tRNA, 7SL, and 5S rRNA. Abnormally high expression of these cellular pol III products may contribute to the ability of EBV to enhance growth potential.

Blotting, Western↗
PubMed2003

Genome-wide location of yeast RNA polymerase III transcription machinery.

RNA polymerase III (Pol III) transcribes a large set of genes encoding small untranslated RNAs like tRNAs, 5S rRNA, U6 snRNA or RPR1 RNA. To get a global view of class III (Pol III-transcribed) genes, the distribution of essential components of Pol III, TFIIIC and TFIIIB was mapped across the yeast genome. During active growth, most class III genes and few additional loci were targeted by TFIIIC, TFIIIB and Pol III, indicating that they were transcriptionally active. SNR52, which encodes a snoRNA, was identified as a new class III gene. During the late growth phase, TFIIIC remained bound to most class III genes while the recruitment of Pol III and, to a lesser extent, of TFIIIB was down regulated. This study fixes a reasonable upper bound to the number of class III genes in yeast and points to a global regulation at the level of Pol III and TFIIIB recruitment.

Base Sequence↗
PubMed1992

The PCF1-1 mutation increases the activity of the transcription factor (TF) IIIB fraction from Saccharomyces cerevisiae.

PCF1-1 is a dominant suppressor of a tRNA gene A block promoter mutation (A19) in Saccharomyces cerevisiae. Transcriptional activation by PCF1-1 was examined in vitro using whole-cell extracts and purified factors derived from mutant and wild-type strains. These experiments show that PCF1 is a general activator of RNA polymerase III (pol III) gene transcription. The transcription of all pol III genes analyzed to date, including type I and numerous type II genes, is increased 3-7 fold in mutant cell extracts. Single round transcription assays indicate that the PCF1-1 mutation increases the number of functional preinitiation complexes and suggest that this is achieved by increasing the intrinsic activity of the encoded product rather than its amount. Point mutations throughout the A block of the sup3-e gene and numerous B block mutations fail to abolish transcriptional activation suggesting that interactions between TFIIIC and the internal promoter are unaffected by PCF1-1. Moreover, TFIIIC purified from the mutant strain is incapable of conferring PCF1-1 transcriptional activity to a reaction in which the remaining components are wild-type. In contrast, the activity of the TFIIIB fraction is increased in PCF1-1 extracts and can reconstitute mutant levels of transcription when added to wild-type TFIIIC and polymerase. We conclude that PCF1 is a component or regulator of TFIIIB.

Base Sequence↗
PubMed1992

The TATA-binding protein participates in TFIIIB assembly on tRNA genes.

The TATA-binding protein TBP has been recently recognized as a general class III transcription factor. Using the gel shift assay to monitor initiation complex assembly on a yeast tRNA gene, we show that TBP is required for the TFIIIC-dependent assembly of TFIIIB. TFIIIB depleted of TBP by a simple chromatographic step does not bind stably to the TFIIIC-tDNA complex. Addition of yeast or human recombinant TBP allows the formation of a TFIIIB-TBP-TFIIIC-tDNA complex. The presence of TBP in the complex was inferred from the effect of anti-TBP antibodies and from the different migration properties of TFIIIB-TBP-tDNA complexes formed with yeast or human TBP.

DNA, Fungal↗
PubMed2001

Comparison of the RNA polymerase III transcription machinery in Schizosaccharomyces pombe, Saccharomyces cerevisiae and human.

Multi-subunit transcription factors (TF) direct RNA polymerase (pol) III to synthesize a variety of essential small transcripts such as tRNAs, 5S rRNA and U6 snRNA. Use by pol III of both TATA-less and TATA-containing promoters, together with progress in the Saccharomyces cerevisiae and human systems towards elucidating the mechanisms of actions of the pol III TFs, provides a paradigm for eukaryotic gene transcription. Human and S.cerevisiae pol III components reveal good general agreement in the arrangement of orthologous TFs that are distributed along tRNA gene control elements, beginning upstream of the transcription initiation site and extending through the 3' terminator element, although some TF subunits have diverged beyond recognition. For this review we have surveyed the Schizosaccharomyces pombe database and identified 26 subunits of pol III and associated TFs that would appear to represent the complete core set of the pol III machinery. We also compile data that indicate in vivo expression and/or function of 18 of the fission yeast proteins. A high degree of homology occurs in pol III, TFIIIB, TFIIIA and the three initiation-related subunits of TFIIIC that are associated with the proximal promoter element, while markedly less homology is apparent in the downstream TFIIIC subunits. The idea that the divergence in downstream TFIIIC subunits is associated with differences in pol III termination-related mechanisms that have been noted in the yeast and human systems but not reviewed previously is also considered.

Amino Acid Sequence↗
PubMed2004

Similarities in transcription factor IIIC subunits that bind to the posterior regions of internal promoters for RNA polymerase III.

BACKGROUND: In eukaryotes, RNA polymerase III (RNAP III) transcribes the genes for small RNAs like tRNAs, 5S rRNA, and several viral RNAs, and short interspersed repetitive elements (SINEs). The genes for these RNAs and SINEs have internal promoters that consist of two regions. These two regions are called the A and B blocks. The multisubunit transcription factor TFIIIC is required for transcription initiation of RNAP III; in transcription of tRNAs, the B-block binding subunit of TFIIIC recognizes a promoter. Although internal promoter sequences are conserved in eukaryotes, no evidence of homology between the B-block binding subunits of vertebrates and yeasts has been reported previously. RESULTS: Here, I reported the results of PSI-BLAST searches using the B-block binding subunits of human and Shizosacchromyces pombe as queries, showing that the same Arabidopsis proteins were hit with low E-values in both searches. Comparison of the convergent iterative alignments obtained by these PSI-BLAST searches revealed that the vertebrate, yeast, and Arabidopsis proteins have similarities in their N-terminal one-third regions. In these regions, there were three domains with conserved sequence similarities, one located in the N-terminal end region. The N-terminal end region of the B-block binding subunit of Saccharomyces cerevisiae is tentatively identified as a HMG box, which is the DNA binding motif. Although I compared the alignment of the N-terminal end regions of the B-block binding subunits, and their homologs, with that of the HMG boxes, it is not clear whether they are related. CONCLUSION: Molecular phylogenetic analyses using the small subunit rRNA and ubiquitous proteins like actin and alpha-tubulin, show that fungi are more closely related to animals than either is to plants. Interestingly, the results obtained in this study show that, with respect to the B-block binding subunits of TFIIICs, animals appear to be evolutionarily closer to plants than to fungi.

Amino Acid Sequence↗
PubMed1993

Mapping regions of yeast transcription factor IIIA required for DNA binding, interaction with transcription factor IIIC, and transcription activity.

TFIIIA, a site-specific DNA-binding protein containing nine zinc finger motifs, is an RNA polymerase III transcription factor responsible for the assembly of a transcription complex on the 5 S RNA gene. We have analyzed the effect of deleting various regions of yeast TFIIIA on its ability to bind to the internal control region of the yeast 5 S RNA gene, to recruit TFIIIC into the TFIIIA.DNA complex and to promote in vitro transcription of the 5 S RNA gene. A truncated polypeptide containing only the three amino-terminal zinc fingers retained the ability to bind specifically to the internal control region of the 5 S RNA gene. The TFIIIA.DNA complex formed with this three zinc finger module was able to recruit TFIIIC but could not promote transcription. An 81-amino acid domain, which interrupts the repeating zinc finger motifs between fingers 8 and 9, was found to be essential for the transcription activity of the protein. Removal of the carboxyl-terminal region of yeast TFIIIA that extends beyond the ninth zinc finger motif had no effect on the in vitro activities of the protein. Extending the carboxyl-terminal deletion to include the ninth zinc finger led to reduced transcription activity. We speculate that the ninth zinc finger serves to anchor the carboxyl-terminal portion of TFIIIA on the 5 S RNA gene, correctly orienting the 81-amino acid domain to serve its role in promoting transcription. Removal of the region of the protein amino-terminal to the first zinc finger motif was without effect. Extending the amino-terminal deletion to include the first zinc finger appeared to decrease the affinity of TFIIIA for DNA without affecting the specificity of the protein-DNA interaction. The resultant TFIIIA.DNA complex was defective in recruiting TFIIIC and did not support transcription.

Amino Acid Sequence↗
PubMed1986

Adenovirus stimulation of transcription by RNA polymerase III: evidence for an E1A-dependent increase in transcription factor IIIC concentration.

Human cells expressing adenovirus E1A proteins transcribe transfected tRNA and adenovirus VAI genes at greater than 10-fold higher levels than uninfected HeLa cells. Here we show that the increased transcription observed in vivo is reflected in the in vitro transcriptional activity of cell extracts. Depletion of E1A protein from these extracts by immunoprecipitation with a monoclonal antibody did not diminish the activity, suggesting that E1A proteins do not stimulate transcription directly. Fractionation of the extracts by chromatography on phosphocellulose suggests that the higher activity of extracts of adenovirus-infected cells was due to increased activity of the transcription factor (TF) which is the limiting component required for specific initiation of tRNA and VAI transcription in extracts of uninfected HeLa cells, i.e. TFIIIC. Template commitment titrations further suggest that the increased TFIIIC activity was due to an increase in the concentration of active TFIIIC. On the basis of these results and recent genetic analyses of early adenovirus promoters, we suggest that E1A proteins stimulate transcription of adenovirus genes indirectly by increasing the effective in vivo concentration of the limiting cellular transcription factors required for their transcription.

Adenovirus Early Proteins↗
PubMed2006

Transcriptional activation of a moderately expressed tRNA gene by a positioned nucleosome.

All of the members of a tRNA1(Gly) multigene family from the mulberry silkworm, Bombyx mori, have identical coding regions and consequently identical internal promoter elements, but are transcribed at different levels. A moderately expressed copy, tRNA1(Gly)-4 from within this multigene family, which was transcribed to 30-50% of the highly transcribed gene copies harboured two typical TATAA box sequences in the 5' upstream region at positions -27 nt and -154 nt with respect to the +1 nt of mature tRNA. Deletion of the distal TATAA sequence at -154 nt brought down the transcription more than 70%, whereas mutation of the proximal element did not affect transcription. tRNA1(Gly)-4 could be readily assembled into chromatin, with a positioned nucleosome in the upstream region, and the assembled nucleosome formed stable complexes with the transcription factors TFIIIC and TFIIIB. Organization of the gene into nucleosomes also enhanced transcription significantly above that of the naked DNA, reaching transcription levels comparable with those of the highly transcribed copies. This nucleosome-mediated enhancement in transcription was absent when the distal TATAA sequences were deleted, whereas mutation of the proximal TATAA element showed no effect. In the absence of the distal TATAA sequences, assembly into the nucleosome inhibited transcription of tRNA1(Gly)-4. TFIIIB bound directly through the distal TATAA sequence at -154 nt and the positioned nucleosome facilitated its interaction with TFIIIC. The direct binding of TFIIIB to the DNA provided anchoring of the factor to the template DNA which conferred a higher stability on the TFIIIB-TFIIIC-DNA complex. We have proposed a novel mechanism for the nucleosome-mediated stimulation of pol III (RNA polymerase III) transcription of tRNA genes, a model not presented previously.

Animals↗
PubMed1991

Two essential components of the Saccharomyces cerevisiae transcription factor TFIIIB: transcription and DNA-binding properties.

RNA polymerase III transcription factor TFIIIB from Saccharomyces cerevisiae contains at least two polypeptides, with apparent masses of 90 and 70 kDa, that were previously identified by photocrosslinking to DNA. It is shown here that TFIIIB can be chromatographically separated into two components, each of which is required for efficient tRNA gene transcription. DNA-protein photocrosslinking experiments show these two components separately contain the 90- and 70-kDa TFIIIB-specific polypeptides. The 70-kDa component forms a heparin-sensitive complex with transcription factor TFIIIC and DNA, stabilizes TFIIIC interaction with the tRNA gene promoter elements, and protects against DNase I digestion in the 3' portion of the upstream DNA sequence that is occupied by TFIIIB. The 90-kDa component of TFIIIB, which only detectably interacts with the TFIIIC-DNA complex when the 70-kDa component is also present, generates the complete DNase I protection pattern of TFIIIB and bestows heparin-insensitivity on the TFIIIB-DNA complex. The resolution of TFIIIB into two functional components further defines the probable steps and interactions involved in the formation of stable transcription complexes.

Chromatography, Ion Exchange↗
PubMed2001

High-mobility-group proteins NHP6A and NHP6B participate in activation of the RNA polymerase III SNR6 gene.

Transcription of yeast class III genes involves the formation of a transcription initiation complex that comprises RNA polymerase III (Pol III) and the general transcription factors TFIIIB and TFIIIC. Using a genetic screen for positive regulators able to compensate for a deficiency in a promoter element of the SNR6 gene, we isolated the NHP6A and NHP6B genes. Here we show that the high-mobility-group proteins NHP6A and NHP6B are required for the efficient transcription of the SNR6 gene both in vivo and in vitro. The transcripts of wild-type and promoter-defective SNR6 genes decreased or became undetectable in an nhp6ADelta nhp6BDelta double-mutant strain, and the protection over the TATA box of the wild-type SNR6 gene was lost in nhp6ADelta nhp6BDelta cells at 37 degrees C. In vitro, NHP6B specifically stimulated the transcription of SNR6 templates up to fivefold in transcription assays using either cell nuclear extracts from nhp6ADelta nhp6BDelta cells or reconstituted transcription systems. Finally, NHP6B activated SNR6 transcription in a TFIIIC-independent assay. These results indicate that besides the general transcription factors TFIIIB and TFIIIC, additional auxillary factors are required for the optimal transcription of at least some specific Pol III genes.

DNA-Binding Proteins↗
PubMed1990

Regulation of RNA polymerase III transcription in response to Simian virus 40 transformation.

Transcription by RNA polymerase III of the B2 family of middle-repetitive elements is activated in response to transformation by a variety of agents, including DNA tumour viruses, RNA tumour viruses and chemical carcinogens. We have investigated the mechanism of activation in SV40-transformed cells and we find that the effect is due to an increase in the activity of the general class III transcription factor TFIIIC, achieved both by an increase in factor abundance and by a change in its phosphorylation state. SV40 transformation also stimulates transcription of other genes by RNA polymerase III but the effect may be balanced by compensatory post-transcriptional changes. TFIIIC may mediate the stimulation of polymerase III transcription by a range of transforming viruses.

Animals↗
PubMed1997

A RNA polymerase III-based two-hybrid system to study RNA polymerase II transcriptional regulators.

In a previous study, we explored the mechanisms of SNR6 gene activation by grafting a heterologous DNA-binding domain, GAL4-(1-147), to various components of the yeast RNA polymerase III transcription system. Here, we demonstrate that a modified SNR6 gene harboring GAL4-binding sites (UAS(G)-SNR6) can be efficiently activated via an intervening, unrelated protein-protein interaction, thus laying the foundations of a RNA polymerase III-based two-hybrid system. In a model system, the interacting proteins recruiting TFIIIC to DNA were PRP21 and PRP9 or PRP21 and PRP11. Mutations affecting the interaction between PRP21 and PRP9, or PRP21 and PRP11 decreased UAS(G)-SNR6 activation level proportionally. RNA polymerase II transcriptional activators, like GAL4, VP16 or p53, fused to GAL4 DNA-binding domain, did not activate the UAS(G)-SNR6 gene. However, GAL4 strongly activated UAS(G)-SNR6 when GAL80, an interacting protein, was fused to TFIIIC. This result indicates that this two-hybrid system can be used to assess the interactions between RNA polymerase II regulatory proteins and their partners.

DNA-Binding Proteins↗