Search

SUMMARYIntegration of transposable elements into the genome is mutagenic. Mechanisms that target integration into relatively safe locations and minimize deleterious consequences for cell fitness have emerged during evolution. In budding yeast, the integration of the Ty1 LTR retrotransposon upstream of RNA polymerase III (Pol III)-transcribed genes requires the interaction between the AC40 subunit of Pol III and Ty1 integrase (IN1). Here we show that the IN1-AC40 interaction involves a short linker sequence in the bipartite nuclear localization signal (bNLS) of IN1. Mutations in this sequence do not impact the frequency of Ty1 retromobility, instead they decrease the recruitment of IN1 to Pol III-transcribed genes and the subsequent integration of Ty1 at these loci. The replacement of Ty5 retrotransposon targeting sequence by the IN1 bNLS induces Ty5 integration into Pol III-transcribed genes. Therefore, the IN1 bNLS is both necessary and sufficient to confer integration site specificity on Ty1 and Ty5 retrotransposons.

Maf1 is a negative regulator of RNA polymerase III (Pol III) in yeast. Maf1-depleted cells manifest elevated tRNA transcription and inability to grow on non-fermentable carbon source, such as glycerol. Using genomic microarray approach, we examined the effect of Maf1 deletion on expression of Pol II-transcribed genes in yeast grown in medium containing glycerol. We found that transcription of FBP1 and PCK1, two major genes controlling gluconeogenesis, was decreased in maf1Δ cells. FBP1 is located on chromosome XII in close proximity to a tRNA-Lys gene. Accordingly we hypothesized that decreased FBP1 mRNA level could be due to the effect of Maf1 on tgm silencing (tRNA gene mediated silencing). Two approaches were used to verify this hypothesis. First, we inactivated tRNA-Lys gene on chromosome XII by inserting a deletion cassette in a control wild type strain and in maf1Δ mutant. Second, we introduced a point mutation in the promoter of the tRNA-Lys gene cloned with the adjacent FBP1 in a plasmid and expressed in fbp1Δ or fbp1Δ maf1Δ cells. The levels of FBP1 mRNA were determined by RT-qPCR in each strain. Although the inactivation of the chromosomal tRNA-Lys gene increased expression of the neighboring FBP1, the mutation preventing transcription of the plasmid-born tRNA-Lys gene had no significant effect on FBP1 transcription. Taken together, those results do not support the concept of tgm silencing of FBP1. Other possible mechanisms are discussed.

We used genome-wide expression analysis in Saccharomyces cerevisiae to explore whether and how the expression of protein-coding, RNA polymerase (Pol) II-transcribed genes is influenced by a decrease in RNA Pol III-dependent transcription. The Pol II transcriptome was characterized in four thermosensitive, slow-growth mutants affected in different components of the RNA Pol III transcription machinery. Unexpectedly, we found only a modest correlation between altered expression of Pol II-transcribed genes and their proximity to class III genes, a result also confirmed by the analysis of single tRNA gene deletants. Instead, the transcriptome of all of the four mutants was characterized by increased expression of genes known to be under the control of the Gcn4p transcriptional activator. Indeed, GCN4 was found to be translationally induced in the mutants, and deleting the GCN4 gene eliminated the response. The Gcn4p-dependent expression changes did not require the Gcn2 protein kinase and could be specifically counteracted by an increased gene dosage of initiator tRNA(Met). Initiator tRNA(Met) depletion thus triggers a GCN4-dependent reprogramming of genome expression in response to decreased Pol III transcription. Such an effect might represent a key element in the coordinated transcriptional response of yeast cells to environmental changes.

International audience; To identify the proteins associated with the RNA polymerase III (Pol III) machinery in exponentially growing yeast cells, we developed our own tandem chromatin affinity purification procedure (TChAP) after in vivo cross-link, allowing a reproducible and good recovery of the protein bait and its associated partners. In contrast to TFIIIA that could only be purified as a free protein, this protocol allows us to capture free Pol III together with Pol III bound on its target genes. Transcription factors, elongation factors, RNA-associated proteins and proteins involved in Pol III biogenesis were identified by mass spectrometry. Interestingly, the presence of all the TFIIIB subunits found associated with Pol III together with the absence of TFIIIC and chromatin factors including histones suggest that DNA-bound Pol III purified using TChAP is mainly engaged in transcription reinitiation.

TFIIIC-dependent assembly of yeast TFIIIB on class III genes unmasks a high avidity of TFIIIB for DNA. TFIIIB contains TATA-binding protein (TBP), TFIIIB90/B", and TFIIIB70/Brf1, which is homologous to TFIIB. Using limited proteolysis, we have found that the COOH terminus of TFIIIB70 (residues 510–596) forms a protease-resistant domain that binds DNA tightly as seen by Southwestern, DNase I footprinting, and gel shift assays. Consistent with a role for this DNA binding activity, preinitiation complexes were formed less efficiently with truncated TFIIIB70 lacking the COOH-terminal domain and displayed an increased sensitivity to heparin. B′ (TFIIIB70 + TBP)·TFIIIC·DNA complexes were also particularly unstable. In addition, TFIIIB·TFIIIC·DNA complexes containing truncated TFIIIB70 were impaired in promoting transcription initiation.

Recent data indicate that the well-defined transcription machinery of RNA polymerase III (Pol III) is probably more complex than commonly thought. In this review, we describe the yeast basal transcription factors of Pol III and their involvements in the transcription cycle. We also present a list of proteins detected on genes transcribed by Pol III (class III genes) that might participate in the transcription process. Surprisingly, several of these proteins are involved in RNA polymerase II transcription. Defining the role of these potential new effectors in Pol III transcription in vivo will be the challenge of the next few years. This article is part of a Special Issue entitled: Transcription by Odd Pols.

While screening for genes that affect the synthesis of yeast snRNPs, we identified a thermosensitive mutant that abolishes the production of a reporter snRNA at the non-permissive temperature. This mutant defines a new gene, named BDF1. In a bdf1-1 strain, the reporter snRNA synthesized before the temperature shift remains stable at the non-permissive temperature. This demonstrates that the BDF1 gene affects the synthesis rather than the stability of the reporter snRNA and suggests that the BDF1 gene encodes a transcription factor. BDF1 is present in single copy on yeast chromosome XII, and is important for normal vegetative growth but not essential for cell viability. bdf1 null mutants share common phenotypes with several mutants affecting general transcription and are defective in snRNA production. BDF1 encodes a protein of 687 amino-acids containing two copies of the bromodomain, a motif also present in other transcription factors as well as a new conserved domain, the ET domain, also present in Drosophila and human proteins.

Yeast transcription factor TFIIIB is a multicomponent factor comprised of the TATA-binding protein TBP and of associated factors TFIIIB70 and B". Epitope-tagged or histidine-tagged TFIIIB70 could be quantitatively removed from TFIIIB by affinity chromatography. TBP and B" (apparent mass 160-200 kDa) could be easily separated by gel filtration or ion-exchange chromatography. While only weak interactions were detected between TBP and B", direct binding of [35S]-labeled TBP to membrane-bound TFIIIB70 could be demonstrated in absence of DNA. On tRNA genes, there was no basal level of transcription in the complete absence of TBP. The two characterized TFIIIB components (recombinant rTFIIIB70 and rTBP) and a fraction cochromatographing with B" activity were found to be required for TFIIIC-independent transcription of the TATA-containing U6 RNA gene in vitro. Therefore, beside the TFIIIC-dependent assembly process, each TFIIIB component must have an essential role in DNA binding or RNA polymerase recruitment.

Saccharomyces cerevisiae transcription factor IIIC/tau is a multisubunit DNA-binding protein that plays key roles in tRNA and 5 S rRNA gene activation. Subunit composition, stoichiometry, and in vivo phosphorylation of TFIIIC/tau factor were investigated using factor prepared from strains carrying modified forms of TFC1, the gene encoding the 95-kDa TFIIIC/tau subunit (tau 95). Using an epitope-tagged TFC1 as well as a TFC1-lacZ fusion, TFIIIC was shown to contain a single 95-kDa subunit, which was localized by electron microscopy into tau A, the A block-binding domain of TFIIIC/tau. Three 35S-labeled polypeptides (at 138, 131, and 91 kDa) coimmunoprecipitated with a tau 95-beta-galactosidase fusion protein. The coprecipitation of the 91-kDa polypeptide makes it a likely subunit of the factor. Immunoprecipitation from 32P-labeled extracts revealed that three of the subunits (138, 131, and 95 kDa), but not the 91-kDa component, are phosphorylated in vivo.

Yeast Sub1 and its human ortholog PC4 display multiple cellular functions in vivo. Sub1/PC4 contains a unique conserved non-specific DNA-binding domain and is involved in distinct DNA-dependent processes including replication, DNA repair and transcription. Sub1/PC4 is a non-histone chromatin-associated protein initially described as a co-activator for RNA polymerase (Pol) II transcription in vitro. Recently, biochemical and genomic studies showed that Sub1 is not restricted to Pol II transcription but is also involved in Pol III transcription revealing a more general role in transcription than anticipated. Sub1/PC4 appears to play a dual (positive and negative) complex role in gene expression and has multiple effects in distinct steps of the transcription cycle, consisting of initiation, elongation, termination and reinitiation. Here, the multiple transcriptional functions of Sub1/PC4 will be reviewed and the recent findings and their possible implications in the understanding of Sub1/PC4 function in transcription will be discussed.

Yeast transcription factor IIIC (TFIIIC) is a multisubunit protein complex that interacts with two control elements of class III promoters called the A and B blocks. Here we describe the gene encoding the 138-kDa subunit (tau 138), which is involved in B-block binding. From the DNA sequence, the open reading frame, interrupted by an intron with an unusual 3' splice acceptor site, is in agreement with all the microsequencing data for peptides within tau 138. TFC3 is a single-copy gene located on chromosome I; it is essential for cell viability as shown by a gene disruption experiment. Epitope-tagging of the TFC3 gene product and DNA binding experiments are consistent with the presence of one copy of tau 138 in TFIIIC-DNA complexes.

Human PC4 and the yeast ortholog Sub1 have multiple functions in RNA polymerase II transcription. Genome-wide mapping revealed that Sub1 is present on Pol III-transcribed genes. Sub1 was found to interact with components of the Pol III transcription system and to stimulate the initiation and reinitiation steps in a system reconstituted with all recombinant factors. Sub1 was required for optimal Pol III gene transcription in exponentially growing cells.

Transcription factor TFIIIC mediates tRNA and 5S RNA gene activation by binding to intragenic promoter elements. The factor from Saccharomyces cerevisiae, also called tau, is a large, multisubunit protein (550-650 kDa) containing two polypeptides that interact directly with DNA encoding tRNA (tDNA). We have obtained peptide sequences from the 95-kDa DNA-binding subunit (tau 95) and cloned the corresponding gene, called TFC1. The gene encodes a polypeptide of calculated Mr 73,500. However, when TFC1 was transcribed and translated in vitro, the gene product comigrated with tau 95 in SDS/polyacrylamide gels. A fusion protein expressed in bacteria was able to prevent the binding of anti-tau 95 antibodies to tau-tDNA complexes. The TFC1 gene is present in single copy on yeast chromosome II and is essential for growth. Spores containing a disrupted gene germinate but only proceed through a few cell divisions before ceasing to grow. The TFC1-encoded protein contains a potential helix-turn-helix structure and an acidic carboxyl-terminal domain, a feature characteristic of some DNA-binding proteins and transcriptional regulators.

RNA polymerase III (Pol III) produces essential components of the biosynthetic machinery, and therefore its activity is tightly coupled with cell growth and metabolism. In the yeast Saccharomyces cerevisiae, Maf1 is the only known global and direct Pol III transcription repressor which mediates numerous stress signals. Here we demonstrate that transcription regulation by Maf1 is not limited to stress but is important for the switch between fermentation and respiration. Under respiratory conditions, Maf1 is activated by dephosphorylation and imported into the nucleus. The transition from a nonfermentable carbon source to that of glucose induces Maf1 phosphorylation and its relocation to the cytoplasm. The absence of Maf1-mediated control of tRNA synthesis impairs cell viability in nonfermentable carbon sources. The respiratory phenotype of maf1-Delta allowed genetic suppression studies to dissect the mechanism of Maf1 action on the Pol III transcription apparatus. Moreover, in cells grown in a nonfermentable carbon source, Maf1 regulates the levels of different tRNAs to various extents. The differences in regulation may contribute to the physiological role of Maf1.

The essential C17 subunit of yeast RNA polymerase (Pol) III interacts with Brf1, a component of TFIIIB, suggesting a role for C17 in the initiation step of transcription. The protein sequence of C17 (encoded by RPC17) is conserved from yeasts to humans. However, mammalian homologues of C17 (named CGRP-RCP) are known to be involved in a signal transduction pathway related to G protein-coupled receptors, not in transcription. In the present work, we first establish that human CGRP-RCP is the genuine orthologue of C17. CGRP-RCP was found to functionally replace C17 in Deltarpc17 yeast cells; the purified mutant Pol III contained CGRP-RCP and had a decreased specific activity but initiated faithfully. Furthermore, CGRP-RCP was identified by mass spectrometry in a highly purified human Pol III preparation. These results suggest that CGRP-RCP has a dual function in mammals. Next, we demonstrate by genetic and biochemical approaches that C17 forms with C25 (encoded by RPC25) a heterodimer akin to Rpb4/Rpb7 in Pol II. C17 and C25 were found to interact genetically in suppression screens and physically in coimmunopurification and two-hybrid experiments. Sequence analysis and molecular modeling indicated that the C17/C25 heterodimer likely adopts a structure similar to that of the archaeal RpoE/RpoF counterpart of the Rpb4/Rpb7 complex. These RNA polymerase subunits appear to have evolved to meet the distinct requirements of the multiple forms of RNA polymerases.

There is limited information on how eukaryotic RNA polymerases (Pol) recognize their cognate preinitiation complex. We have characterized a polypeptide copurifying with yeast Pol III. This protein, C17, was found to be homologous to a mammalian protein described as a hormone receptor. Deletion of the corresponding gene, RPC17, was lethal and its regulated extinction caused a selective defect in transcription of class III genes in vivo. Two-hybrid and coimmunoprecipitation experiments indicated that C17 interacts with two Pol III subunits, one of which, C31, is important for the initiation reaction. C17 also interacted with TFIIIB70, the TFIIB-related component of TFIIIB. The interaction domain was found to be in the N-terminal, TFIIB-like half of TFIIIB70, downstream of the zinc ribbon and first imperfect repeat. Although Pol II similarly interacts with TFIIB, it is notable that C17 has no similarity to any Pol II subunit. The data indicate that C17 is a novel specific subunit of Pol III which participates together with C34 in the recruitment of Pol III by the preinitiation complex.

The primary step in tRNA gene activation is the binding of TFIIIC to the A and B blocks of the intragenic promoter. Its main function is then to assemble the initiation factor TFIIIB upstream of the transcription start site (30). Yeast TFIIIC (τ factor) is a multisubunit protein of ca. 600 kDa organized in two large globular domains τA and τB of similar size and mass (10-nm diameter and ca. 300 kDa), each interacting with one promoter element, as visualized by electron microscopy (17, 53). Binding of τB to the B block is predominant and favors the binding of τA to the A block (4). TFIIIC-DNA interaction displays a remarkable adaptability to the variable A-B distances found in different tRNA genes (3). Affinity-purified Saccharomyces cerevisiae TFIIIC comprises six polypeptides of 138, 131, 95, 91, 60, and 55 kDa (6, 19, 47, 59). Gene cloning, protein-DNA cross-linking, mutagenesis, and protein-protein interaction studies provided a global view of the location and role of several TFIIIC subunits within the TFIIIC-DNA complex. τ138 and τ91 subunits reside in the τB domain and cooperate in downstream DNA binding (1, 10, 36, 37); τ95 and τ55 interact physically, belong to the τA domain, and are thought to participate in A block binding (7, 17, 40, 48, 59). τ131 (42) stands as the TFIIIC subunit responsible for TFIIIB assembly based on genetic evidence (49, 61), its upstream location (7), and its interaction with two TFIIIB components (14, 33, 51). TFIIIB is not a stable molecular entity like TFIIIC. It can be resolved chromatographically into two fractions named B′ and B" (29). B′ comprises TATA-binding protein (TBP) and the TFIIB-related factor TFIIIB70/Brf1 (12, 16, 31, 39), while B" contains TFIIIB90 (32, 50, 51). The TFIIIC-dependent TFIIIB assembly on TATA-less class III genes is a multistep pathway that could be decomposed in vitro (29, 31) and reconstituted with recombinant TFIIIB components (32, 51). The order of interaction is TFIIIB70, then TBP, and then B", as shown by gel retardation and DNA photo-cross-linking (31). TBP stabilizes the weak interaction between TFIIIB70 and the TFIIIC-DNA complex but the complete upstream footprint and the characteristic stability of the TFIIIB-DNA complex requires the recruitment of B"/TFIIIB90 (29, 31). A cascade of conformational rearrangements at the protein and DNA levels are accompanying these assembly steps, as evidenced by successive changes in the accessibility of TFIIIB70, TBP, and τ131 to site-specific DNA cross-linking (31), by the DNA bending induced upon TFIIIB binding (11, 38, 46), and by the presence of a cryptic DNA binding domain in TFIIIB70 (24). τ131 appears to play the major role in positioning TFIIIB since it is the only TFIIIC subunit accessible to DNA cross-linking upstream of the start site (5, 7) and found to interact with TFIIIB70 (14, 33) and TFIIIB90 (51). TFIIIB can effect its own assembly onto the TATA-containing SNR6 gene through the interaction of TBP with the strong TATA box (27, 43, 45). Interestingly, Whitehall et al. (60) found that TBP could not discern the polarity of the TATA element and directed TFIIIB assembly in two orientations. However, in contrast to the TATA-dependent assembly, TFIIIC placed TFIIIB in the correct orientation. Since no TFIIIC component was known to interact with TBP, it was presumed that the unidirectional binding of TBP to the TATA box is dictated by the oriented interaction of TFIIIB70 with τ131 (60). In the present work we have completed the characterization of TFIIIC components by cloning a yeast gene, named TFC8, that encodes the 60-kDa polypeptide. We present genetic and biochemical evidence that this component, named τ60, resides at least in part within the τB domain and participates in TFIIIB recruitment via TBP binding.

In eucaryotic cells, the transcription of a variety of small genes is conducted by RNA polymerase III (PolIII) and requires several auxiliary factors. For yeast tRNA gene (tDNA) activation, preinitiation complexes are assembled in a defined order within and upstream of the transcription unit (18, 27, 52). Transcription factor IIIC (TFIIIC) plays a primary role in this multistep complex assembly by binding to the intragenic promoter elements of tRNA genes (the A and B blocks). Yeast TFIIIC is a remarkably large multisubunit factor made of two protein subassemblies, named τA and τB, that have distinct DNA binding properties, that can be visualized by electron microscopy in a free or DNA-bound form (46), and that can be cleaved by limited proteolysis (37). The τB domain binds tightly to the B block (37) and has been shown to display all the properties of enhancer binding proteins (11). Binding of the τA domain to the A block is weaker and mostly B block dependent. Once bound, TFIIIC promotes the binding of transcription factor IIIB (TFIIIB) upstream of the transcription start site (6, 26, 28, 31). The process is similar for yeast 5S RNA gene activation, except that TFIIIC assembly is dependent upon the binding of transcription factor IIIA (TFIIIA) to the internal promoter sequence. TFIIIB by itself does not bind detectably to TATA-less PolIII genes but, once assembled via TFIIIC, interacts intimately with DNA and is sufficient, at least in the yeast system, for directing accurate initiation by PolIII during multiple rounds of transcription in vitro (28, 31). Hence, TFIIIB is the initiation factor required for the activation of all PolIII genes, whereas TFIIIC and TFIIIA act as assembly factors. However, it has been shown that TFIIIC is a multifunctional protein, involved not only in promoter recognition and TFIIIB recruitment but also in the displacement of nucleosomes to relieve the repression of transcription by chromatin (10). The molecular structure of yeast TFIIIB and TFIIIC has been much investigated. Yeast TFIIIB comprises three components: TBP, the TATA box binding protein, which is also required for transcription by PolI and PolII, and two additional polypeptides, TFIIIB70/BRF1 and TFIIIB90/B", first identified by protein-DNA cross-linking (6). Together with TBP, TFIIIB70 is able to bind to TFIIIC-tDNA complexes (29). The resulting complex becomes competent to recruit PolIII after the assembly of TFIIIB90 (29, 30). Purified yeast TFIIIC comprises six polypeptides, of 138, 131, 95, 91, 60, and 55 kDa (5, 16, 43). Purification of TFIIIC to near homogeneity, protein-DNA cross-linking (5, 9, 16), and coimmunoprecipitation experiments (13, 44) suggested that the four largest polypeptides were subunits of TFIIIC, a suggestion which was confirmed by gene cloning (2, 34, 36, 44, 48). The 138- and 95-kDa components (τ138 and τ95), located in τB and τA, respectively, are thought to be DNA binding subunits, since they could be specifically cross-linked to a tDNA probe and were mapped at the level of the B block and the A block, respectively (5, 9, 13, 16). τ91 was recently shown to cooperate with τ138 for TFIIIC-tDNA binding (2) and was mapped at the most 3′ location of TFIIIC-5S RNA gene complexes (9). τ131 stands as the TFIIIB-assembling subunit based on its upstream gene location, shown by protein-DNA cross-linking (5, 6), and its direct interaction with both TFIIIB70 and TFIIIB90 (12, 32, 45). On the other hand, little is known about the smallest polypeptides, of 60 and 55 kDa, which reproducibly copurify with yeast TFIIIC activity. Both proteins were found among the six polypeptides isolated from TFIIIC-tDNA complexes (16). A 55-kDa polypeptide was located by photo-cross-linking experiments together with τ95, on opposite sides of the DNA helix, in the vicinity of the A block of tRNA genes (5, 6). The 60-kDa polypeptide was not cross-linked to DNA. We report here the cloning and identification of TFC7, an essential gene encoding the 55-kDa subunit of TFIIIC. Analysis of deletion mutants showed that only the C-terminal half of τ55 is necessary for TFIIIC transcriptional activity. We found that τ55 interacts with τ95 and that these two subunits are present in at least two distinct protein complexes.

Transcription factor IIIC (TFIIIC) (or t) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, t91, present in affinity-purified TFIIIC. t91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of t91 was established by the following biochemical and genetic evidence. Antibodies to t91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to t91; and a point mutation in TFC6 (t91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349E mutant affected in the B block binding subunit (t138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that t91 cooperates with t138 for DNA binding. Recombinant t91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA. Transcription of class III genes involves multiple interactions between promoter elements, auxiliary transcription factors, and RNA polymerase III (Pol III). Prototypical class III genes, like tRNA and 5S RNA genes, have intragenic promoter elements or internal control regions, differing in this way from class I and class II genes. The internal control regions of

Publisher Summary RNA polymerase III is specifically recruited at the transcription start sites via a cascade of protein-protein-DNA interactions involving various transcription factors. Remarkably, complex assemblies are formed that involve more than 25 polypeptides and cover the entire transcription units. The transcriptional components and the process of transcription complex formation on various class III genes have been best analyzed in Saccharomyces cerevisiae. This chapter describes the purification of yeast RNA polymerase III and of yeast factors TFIIIA (transcription factor IIIA), TFIIIB, and TFIIIC. RNA polymerase III and TFIIIC can be purified to near homogeneity as stable multisubunit assemblies. In contrast, TFIIIB can be easily dissociated into three components (TBP, TFIIIB, and B"). TFIIIA, TFIIIB and TBP can be obtained in active form as recombinant proteins made in Escherichia coli . Twenty components of the yeast class III transcription machinery have already been cloned and mutagenized.

Transcription of yeast class III genes requires the sequential assembly of the general transcription factors TFIIIC and TFIIIB, and of RNA polymerase III, into an initiation complex composed of at least 25 polypeptides. The 70-kDa subunit of TFIIIB (TFIIIB70) is central in this network of interactions as it contacts both TATA-binding protein and a subunit of polymerase III. We show here that the TATA-binding protein interacts with the carboxyl-terminal part of TFIIIB70. TFIIIB70 also contacts TFIIIC (factor τ) via its τ131 subunit. The protein domains of τ131 and TFIIIB70 involved in this interaction, either positively or negatively, were mapped using the two-hybrid system. We provide evidence that intramolecular interactions mask functional domains in both polypeptides.

A yeast chimeric RNA polymerase III transcription system was constructed to explore the ordered, multistep process of gene activation in vivo. A promoter-deficient U6 RNA gene harboring GAL4-binding sites could be reactivated by fusing the GAL4 DNA-binding domain to components of the general transcription factor TFIIIC (tau) or TFIIIB. Expression of chimeric tau 138 or tau 131 (but not tau 95) subunits activated transcription from GAL4-binding sites located at various positions, including upstream of or within the gene. The function(s) of the B block binding domain of TFIIIC was provided by the fused GAL4-(1-147) domain. The GAL4-(1-147)-TFIIIB70 fusion protein acted at a distance like an activator of transcription. In contrast, none of the 10 different GAL4-(1-147)-polymerase subunit fusions was able to induce transcription, suggesting that RNA polymerase recruitment is not sufficient to initiate transcription.

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.