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RNA polymerase III (RNAPIII) synthesizes tRNA, 5S RNA, U6 snRNA, and other small RNAs. The structure of yeast RNAPIII, determined at 17 A resolution by cryo-electron microscopy and single-particle analysis, reveals a hand-like shape typical of RNA polymerases. Compared to RNAPII, RNAPIII is characterized by a bulkier stalk and by prominent features extending from the DNA binding cleft. We attribute the latter primarily to five RNAPIII-specific subunits, present as two distinct subcomplexes (C82/C34/C31 and C53/C37). Antibody labeling experiments localize the C82/C34/C31 subcomplex to the clamp side of the DNA binding cleft, consistent with its known role in transcription initiation. The C53/C37 subcomplex appears to be situated across the cleft, near the presumed location of downstream DNA, accounting for its role in transcription termination. Our structure rationalizes available mutagenesis and biochemical data and provides insights into RNAPIII-mediated transcription.

Roger Kornberg, 59 ans, Americain, ne en 1947 a Saint-Louis (Missouri, Etats-Unis), est professeur de medecine a l’Universite de Stanford (Californie, Etats-Unis). Il a 12 ans lorsqu’il accompagne son pere Arthur Kornberg a Stockholm, ou celui-ci se voit remettre le Prix Nobel de medecine. C’est a Stanford qu’il a fait l’essentiel de ses etudes et de sa carriere, si l’on excepte une parenthese a l’universite britannique de Cambridge entre 1978 et 1984. Roger Kornberg avait auparavant recu plusieurs distinctions dont le Prix Charles Leopold Mayer en 2002, ainsi que le Prix Gairdner en 1995.

Yeast RNA polymerase III is recruited upon binding of subcomplexes tauA and tauB of transcription factor IIIC (TFIIIC) to the A and B blocks of tRNA gene promoters. The tauB subcomplex consists of subunits tau60, tau91, and tau138. We determined the 3.2 A crystal structure of tau60 bound to a large C-terminal fragment of tau91 (Deltatau91). Deltatau91 protein contains a seven-bladed propeller preceded by an N-terminal extension, whereas tau60 contains a structurally homologous propeller followed by a C-terminal domain with a novel alpha/beta fold. The two propeller domains do not have any detectable DNA binding activity and mediate heterodimer formation that may serve as scaffold for tau138 assembly. We show that the C-terminal tau60 domain interacts with the TATA binding protein (TBP). Recombinant tauB recruits TBP and stimulates TFIIIB-directed transcription on a TATA box containing tRNA gene, implying a combined contribution of tauA and tauB to preinitiation complex formation.

Regulation of ribosome biogenesis is a key element of cell biology, not only because ribosomes are directly required for growth, but also because ribosome production monopolizes nearly 80% of the global transcriptional activity in rapidly growing yeast cells. These observations underscore the need for a tight regulation of ribosome synthesis in response to environmental conditions. In eukaryotic cells, ribosome synthesis involves the activities of the three nuclear RNA polymerases (Pol). Although postulated, there is no clear evidence indicating whether the maintenance of an equimolar supply of ribosomal components reflects communication between the nuclear transcriptional machineries. Here, by constructing a yeast strain expressing a Pol I that remains constitutively competent for the initiation of transcription under stress conditions, we demonstrate that derepression of Pol I transcription leads to a derepression of Pol II transcription that is restricted to the genes encoding ribosomal proteins. Furthermore, we show that the level of 5S rRNA, synthesized by Pol III, is deregulated concomitantly with Pol I transcription. Altogether, these results indicate that a partial derepression of Pol I activity drives an abnormal accumulation of all ribosomal components, highlighting the critical role of the regulation of Pol I activity within the control of ribosome biogenesis.

We report genome-wide analyses that establish Maf1 as a general and direct repressor of yeast RNA polymerase (Pol) III transcription. Chromatin immunoprecipitation (ChIP) coupled to microarray hybridization experiments showed an increased association of Maf1 to Pol III-transcribed genes under repressing condition (rapamycin treatment) correlated with a dissociation of Brf1 and Pol III. Maf1 can exist in various phosphorylation states and interacts with Pol III in a dephosphorylated state. The largest subunit of Pol III, C160, was identified as a target of Maf1. Under repressing conditions, Maf1 is dephosphorylated and accumulates in the nucleus, and Pol III-Maf1 interaction increases. Mutations in protein phosphatase type 2A (PP2A) catalytic subunit-encoding genes prevented rapamycin-induced Maf1 dephosphorylation, its nuclear accumulation, and repression of Pol III transcription. The results indicate that Pol III transcription can be globally and rapidly downregulated via dephosphorylation and relocation of a general negative cofactor.

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.

Gene transcription is repetitive, enabling the synthesis of multiple copies of identical RNA molecules from the same template. The cyclic process of RNA synthesis from active genes, referred to as transcription reinitiation, contributes significantly to the level of RNAs in living cells. Contrary to the perception that multiple transcription cycles are a mere iteration of mechanistically identical steps, a large body of evidence indicates that, in most transcription systems, reinitiation involves highly specific and regulated pathways. These pathways influence the availability for reinitiation of template DNA and/or transcription proteins, and represent an important yet poorly characterized aspect of gene regulation.

A43, an essential subunit of yeast RNA polymerase I (pol I), interacts with Rrn3, a class I general transcription factor required for rDNA transcription. The pol I–Rrn3 complex is the only form of enzyme competent for promoter-dependent transcription initiation. In this paper, using biochemical and genetic approaches, we demonstrate that the A43 polypeptide forms a stable heterodimer with the A14 pol I subunit and interacts with the common ABC23 subunit, the yeast counterpart of the ω subunit of bacterial RNA polymerase. We show by immunoelectronic microscopy that A43, ABC23, and A14 colocalize in the three-dimensional structure of the pol I, and we demonstrate that the presence of A43 is required for the stabilization of both A14 and ABC23 within the pol I. Because the N-terminal half of A43 is clearly related to the pol II Rpb7 subunit, we propose that the A43–A14 pair is likely the pol I counterpart of the Rpb7–Rpb4 heterodimer, although A14 distinguishes from Rpb4 by specific sequence and structure features. This hypothesis, combined with our structural data, suggests a new localization of Rpb7–Rpb4 subunits in the three-dimensional structure of yeast pol II.

Genome-wide studies have recently revealed the unexpected complexity of the genetic response to apparently simple physiological changes. Here, we show that when yeast cells are exposed to Cd(2+), most of the sulfur assimilated by the cells is converted into glutathione, a thiol-metabolite essential for detoxification. Cells adapt to this vital metabolite requirement by modifying globally their proteome to reduce the production of abundant sulfur-rich proteins. In particular, some abundant glycolytic enzymes are replaced by sulfur-depleted isozymes. This global change in protein expression allows an overall sulfur amino acid saving of 30%. This proteomic adaptation is essentially regulated at the mRNA level. The main transcriptional activator of the sulfate assimilation pathway, Met4p, plays an essential role in this sulfur-sparing response.

RNA polymerase I (Pol I) is dedicated to transcription of the large ribosomal DNA (rDNA). The mechanism of Pol I recruitment onto rDNA promoters is poorly understood. Here we present evidence that subunit A43 of Pol I interacts with transcription factor Rrn3: conditional mutations in A43 were found to disrupt the transcriptionally competent Pol I-Rrn3 complex, the two proteins formed a stable complex when co-expressed in Escherichia coli, overexpression of Rrn3 suppressed the mutant phenotype, and A43 and Rrn3 mutants showed synthetic lethality. Consistently, immunoelectron microscopy data showed that A43 and Rrn3 co-localize within the Pol I-Rrn3 complex. Rrn3 has several protein partners: a two-hybrid screen identified the C-terminus of subunit Rrn6 of the core factor as a Rrn3 contact, an interaction supported in vitro by affinity chromatography. Our results suggest that Rrn3 plays a central role in Pol I recruitment to rDNA promoters by bridging the enzyme to the core factor. The existence of mammalian orthologues of A43 and Rrn3 suggests evolutionary conservation of the molecular mechanisms underlying rDNA transcription in eukaryotes.

Yeast TFIIIC mediates transcription of class III genes by promoting the assembly of a stable TFIIIB-DNA complex that is sufficient for RNA polymerase III recruitment and function. Unexpectedly, we found an interaction in vivo and in vitro between the TFIIIB-recruiting subunit of TFIIIC, tau131, and ABC10alpha, a small essential subunit common to the three forms of nuclear RNA polymerases. This interaction was mapped to the C-terminal region of ABC10alpha. A thermosensitive mutation in the C terminus region of ABC10alpha (rpc10-30) was found to be selectively suppressed by overexpression of a mutant form of tau131 (tau131-DeltaTPR2) that lacks the second TPR repeat. Remarkably, the rpc10-30 mutation weakened the ABC10alpha-tau131 interaction, and the suppressive mutation, tau131-DeltaTPR2 increased the interaction between the two proteins in the two-hybrid assay. These results point to the potential importance of a functional contact between TFIIIC and RNA polymerase III.

The yeast TFIIIB transcription factor is composed of three components, TBP, TFIIIB90 or B″, and TFIIIB70 or BRF. TFIIIB70 is a pivotal component since it interacts with TBP, TFIIIC and RNA polymerase III (pol III). In order to better understand the role of TFIIIB70, we mutagenized extensively three evolutionary conserved motifs of its pol III-specific C-terminal extension. Conditional mutations lying in conserved regions II and III were obtained, some of which altered the interaction with the C34 subunit of pol III and were co-lethal with rpc34 mutations. Two conditional mutations in region II impaired the interaction with TBP and were suppressed by its overexpression. The pattern of suppression of the strongest mutation by overexpression of various mutant TBP, suggested a contact between TBP-R220 and TFIIIB70-D464 residues in vivo. As expected, this TFIIIB70 mutation impaired the assembly of TFIIIB·TFIIIC·DNA complexes and affected in vitro transcription of the SUP4 tRNA gene. Our results underscore the important role of region II of TFIIIB70 in pre-initiation as well as transcription complex assembly via C34 and TBP binding.

Budding yeast RNA polymerase III (Pol III) contains a small, essential subunit, named C11, that is conserved in humans and shows a strong homology to TFIIS. A mutant Pol III, heterocomplemented withSchizosaccharomyces pombe C11, was affected in transcription termination in vivo. A purified form of the enzyme (Pol IIIΔ), deprived of C11 subunit, initiated properly but ignored pause sites and was defective in termination. Remarkably, Pol III Δ lacked the intrinsic RNA cleavage activity of complete Pol III. In vitro reconstitution experiments demonstrated that Pol III RNA cleavage activity is mediated by C11. Mutagenesis in C11 of two conserved residues, which are critical for the TFIIS-dependent cleavage activity of Pol II, is lethal. Immunoelectron microscopy data suggested that C11 is localized on the mobile thumb-like stalk of the polymerase. We propose that C11 allows the enzyme to switch between an RNA elongation and RNA cleavage mode and that the essential role of the Pol III RNA cleavage activity is to remove the kinetic barriers to the termination process. The integration of TFIIS function into a specific Pol III subunit may stem from the opposite requirements of Pol III and Pol II in terms of transcript length and termination efficiency.

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.

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.

The yeast RNA polymerase I is a multimeric complex composed of 14 distinct subunits, 5 of which are shared by the three forms of nuclear RNA polymerase. The reasons for this structural complexity are still largely unknown. Isolation of an inactive form of RNA Pol I lacking the A43, ABC23, and A14 subunits (RNA Pol I delta) allowed us to investigate the function of the shared subunit ABC23 by in vitro reconstitution experiments. Addition of recombinant ABC23 alone to the RNA Pol I delta reactivated the enzyme to up to 50% of the wild-type enzyme activity. The recombinant subunit was stably and stoichiometrically reassociated within the enzymatic complex. ABC23 was found to be required for the formation of the first phosphodiester bond, but it was not involved in DNA binding by RNA Pol I, as shown by gel retardation and surface plasmon resonance experiments, and did not recycle during transcription. Electron microscopic visualization and electrophoretic analysis of the subunit depleted and reactivated forms of the enzyme indicate that binding of ABC23 caused a major conformational change leading to a transcriptionally competent enzyme. Altogether, our results demonstrate that the ABC23 subunit is required for the structural and functional integrity of RNA Pol I and thus should be considered as part of the core enzyme.

A34.5, a phosphoprotein copurifying with RNA polymerase I (Pol I), lacks homology to any component of the Pol II or Pol III transcription complexes. Cells devoid of A34.5 hardly affect growth and rRNA synthesis and generate a catalytically active but structurally modified enzyme also lacking subunit A49 upon in vitro purification. Other Pol I-specific subunits (A49, A14, and A12.2) are nonessential for growth at 30 degrees C but are essential (A49 and A12.2) or helpful (A14) at 25 or 37 degrees C. Triple mutants without A34.5, A49, and A12.2 are viable, but inactivating any of these subunits together with A14 is lethal. Lethality is rescued by expressing pre-rRNA from a Pol II-specific promoter, demonstrating that these subunits are collectively essential but individually dispensable for rRNA synthesis. A14 and A34.5 single deletions affect the subunit composition of the purified enzyme in pleiotropic but nonoverlapping ways which, if accumulated in the double mutants, provide a structural explanation for their strict synthetic lethality. A34.5 (but not A14) becomes quasi-essential in strains lacking DNA topoisomerase I, suggesting a specific role of this subunit in helping Pol I to overcome the topological constraints imposed on ribosomal DNA by transcription.

RNA polymerase I of Saccharomyces cerevisiae is composed of 14 subunits. All of the corresponding genes have been cloned with the exception of the RPA14 gene encoding A14, a specific polypeptide of this enzyme. We report the cloning and the characterization of RPA14. The A14 polypeptide was separated from the other RNA polymerase I subunits by reverse-phase high pressure liquid chromatography and digested with proteinase K. Based on the amino acid sequence of one of the resulting peptides, a degenerate oligonucleotide was synthesized and used to isolate the RPA14 gene from a yeast subgenomic DNA library. RPA14 is a single copy gene that maps to chromosome IV and is flanked by CYP1 and HOM2. Disruption of RPA14 is not lethal, but growth of the rpa14::URA3 mutant strain is impaired at 37 and 38°C. RNA polymerase I was purified from the rpa14::URA3 strain. After two purification steps, the enzyme did not contain the subunits A14, ABC23, and A43. This form of the enzyme was not active in a nonspecific in vitro transcription assay. These results demonstrate that A14 is a genuine subunit of RNA polymerase I and suggest that A14 plays a role in the stability of a subgroup of subunits.

The C31 subunit belongs to a complex of three subunits (C31, C34 and C82) specific to RNA polymerase (pol) III that have no counterparts in other RNA polymerases. This complex is thought to play a role in transcription initiation since it interacts with the general initiation factor TFIIIB via subunit C34. We have obtained a conditional mutation of pol III by partially deleting the acidic C-terminus of the C31 subunit. A Saccharomyces cerevisiae strain carrying this truncated C31 subunit is impaired in in vivo transcription of tRNAs and failed to grow at 37 degrees C. This conditional growth phenotype was suppressed by overexpression of the gene coding for the largest subunit of pol III (C160), suggesting an interaction between C160 and C31. The mutant pol III enzyme transcribed non-specific templates at wild-type rates in vitro, but was impaired in its capacity to transcribe tRNA genes in the presence of general initiation factors. Transcription initiation, but not termination or recycling of the enzyme, was affected in the mutant, suggesting that it could be altered on interaction with initiation factors or on the formation of the open complex. Interestingly, the C-terminal deletion was also suppressed by a high gene dosage of the DED1 gene encoding a putative helicase.

Pioneering studies on eukaryotic transcription were undertaken with the bacterial system in mind. Will the bacterial paradigm apply to eukaryotes? Are there promoter sites scattered in the eukaryotic genome, and sigma-like proteins? Why three forms of RNA polymerase in eukaryotic cells? Why are they structurally so complex, in particular RNA polymerases I and III, compared to the bacterial enzyme? These questions and others that were raised along the way are evoked in this short historical survey of odd RNA polymerases studies, with some emphasis on the contribution of these studies to our global understanding of eukaryotic transcription systems. This article is part of a Special Issue entitled: Transcription by Odd Pols.

We report the characterization of a mutation affecting tau 138, the largest subunit of yeast transcription factor IIIC (TFIIIC). A previously described thermosensitive mutation (tsv115), tightly linked to the centromere of chromosome I (Harris, S.D., and Pringle, J.R. (1991) Genetics 127, 279-285) is shown to lie in the TFC3 gene which encodes tau 138. The tau 138 subunit carrying this mutation bears a single substitution of Glu for Gly at position 349 (G349E). In extracts from mutant cells, both the level of TFIIIC and its affinity for tDNA were found to be reduced. The tDNA binding activity of mutant TFIIIC protein was very sensitive to mild heat treatments, and TFIIIC-DNA interaction was inhibited at moderate salt concentrations, as evidenced by gel shift assays. In addition, the tsv115 mutation affected 5 S RNA synthesis in vitro, suggesting that the tau 138 subunit also plays a role in recognition of the TFIIIA-5 S DNA complex. Multicopy suppressors of the TFIIIC defect were sought to reveal components participating in TFIIIC function. One class of suppressors encodes known components of the transcription machinery: two TFIIIC subunits, tau 95 and tau 131, the 70-kDa subunit of TFIIIB, TBP, and a shared subunit of RNA polymerase (pol) I, II, and III, ABC10 alpha; it also includes genes potentially related to pol III function, such as SRP40 which also suppresses a mutation in a subunit shared by RNA polymerases I and III. A second class of suppressors is not involved in transcription but alleviates the main physiological defects of mutant cells. It includes RPR1 and NOP1, required for the maturation of pre-tRNA and pre-rRNA, respectively.

The FHL1 gene was isolated by screening for high-copy-number suppressors of conditional RNA polymerase III mutations. This gene is unique on the yeast genome and was located close to RPC40 and PRE2 on the right arm of chromosome XVI. It codes for a 936-amino-acid protein containing a domain similar to the fork head DNA-binding domain, initially found in the developmental fork head protein of Drosophila melanogaster and in the HNF-3 family of hepatocyte mammalian transcription factors. Null mutations caused a severe reduction in growth rate and a lower rRNA content that resulted from defective rRNA processing. There was no detectable effect on mRNA splicing. Thus, the Fhl1p protein plays a key role in the control of rRNA processing, presumably by acting as a transcriptional regulator of genes specifically involved in that process. Moreover, mutants carrying the RNA polymerase III mutations were slightly defective in rRNA processing. This accounts for the isolation of FHL1 as a dosage-dependent suppressor and suggests that rRNA processing depends on a still-unidentified RNA polymerase III transcript.

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.

The AC40 and AC19 subunits (encoded by RPC40 and RPC19) are shared by yeast RNA polymerases I and III and have a local sequence similarity to prokaryotic alpha subunits. Mutational analysis of the corresponding "alpha motif" indicated that its integrity is essential on AC40 subunit but is not essential on AC19 subunit. By applying the two-hybrid method, these two polypeptides were shown to associate in vivo. Extragenic suppression of rpc19 and rpc40 mutations confirmed that AC19 and AC40 subunits interact with each other in vivo and revealed an interaction with ABC10 beta subunit [encoded by RPB10; Woychick, N. A. & Young, R.A. (1990) J. Biol. Chem. 265, 17816-17819], one of the five polypeptides common to all three nuclear RNA polymerases. A correction of the RPB10 sequence showed that ABC10 beta subunit is a 70-amino acid polypeptide, as confirmed by peptide microsequencing. These results suggest that the assembly of RNA polymerase I and III requires the association of ABC10 beta subunit with an AC19/AC40 heterodimer.

The selenocysteine tRNA(Sec) gene possesses two external promoter elements, one of which is constituted by a strong TATA box. Point mutant analysis performed in this study led to the conclusion that the functional TATA promoter actually encompasses the sequence -34 GGGTATAAAAGG-23. Individual changes at T-31 do not affect transcription much. Position T-29 is less permissive to mutation since transversion to a G, for example, is less well tolerated than at T-31. Interestingly, a double point mutation, converting GG(-33/-32) to TT, causes abrogation of transcription in vivo and severe reduction of transcription in vitro with human TBP. Therefore, data obtained underscore the fact that, in the Xenopus tRNA(Sec), these two Gs are an integral part of the TATA promoter. Gel retardation experiments indicate that the GG to TT substitution, which led human TBP to lose its ability to support efficient transcription in vitro, correlates with the appearance of an altered pattern of retarded complexes. Altogether, the data presented in this report support a model in which TBP interacts directly with the TATA element of the tRNA(Sec) gene, in contrast to the type of interaction proposed for classical TATA-less tRNA genes.

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.

RPC53 is shown to be an essential gene encoding the C53 subunit specifically associated with yeast RNA polymerase C (III). Temperature-sensitive rpc53 mutants were generated and showed a rapid inhibition of tRNA synthesis after transfer to the restrictive temperature. Unexpectedly, the rpc53 mutants preferentially arrested their cell division in the G1 phase as large, round, unbudded cells. The RPC53 DNA sequence is predicted to code for a hydrophilic M(r)-46,916 protein enriched in charged amino acid residues. The carboxy-terminal 136 amino acids of C53 are significantly similar (25% identical amino acid residues) to the same region of the human BN51 protein. The BN51 cDNA was originally isolated by its ability to complement a temperature-sensitive hamster cell mutant that undergoes a G1 cell division arrest, as is true for the rpc53 mutants.

The gene encoding the 49-kDa subunit of RNA polymerase A in Saccharomyces cerevisiae has been identified by formation of a hybrid enzyme between the S. cerevisiae A49 subunit and Saccharomyces douglasii subunits based on a polymorphism existing between the subunits of RNA polymerase A in these two species. The sequence of the gene reveals a basic protein with an unusually high lysine content, which may account for the affinity for DNA shown by the subunit. No appreciable homology with any polymerase subunits, enzymes, or transcription factors is found. Complete deletion of the single-copy RPA49 gene leads to viable but slowly growing colonies. Insertion of the HIS3 gene halfway into the RPA49 coding region results in synthesis of a truncated A49 subunit that is incorporated into the polymerase. The truncated and wild-type subunits compete equally for assembly in the heterozygous diploid, although the wild type is phenotypically dominant.

RNA polymerase C (III) promotes the transcription of tRNA and 5S RNA genes. In Saccharomyces cerevisiae, the enzyme is composed of 15 subunits, ranging from 160 to about 10 kDa. Here we report the cloning of the gene encoding the 82-kDa subunit, RPC82. It maps as a single-copy gene on chromosome XVI. The UCR2 gene was found in the opposite orientation only 340 bp upstream of the RPC82 start codon, and the end of the SKI3 coding sequence was found only 117 bp downstream of the RPC82 stop codon. The RPC82 gene encodes a protein with a predicted M(r) of 73,984, having no strong sequence similarity to other known proteins. Disruption of the RPC82 gene was lethal. An rpc82 temperature-sensitive mutant, constructed by in vitro mutagenesis of the gene, showed a deficient rate of tRNA relative to rRNA synthesis. Of eight RNA polymerase C genes tested, only the RPC31 gene on a multicopy plasmid was capable of suppressing the rpc82(Ts) defect, suggesting an interaction between the polymerase C 82-kDa and 31-kDa subunits. A group of RNA polymerase C-specific subunits are proposed to form a substructure of the enzyme.

The RPC34 gene of Saccharomyces cerevisiae was cloned by immunological screening, using antibodies raised against the C34 polypeptide of the RNA polymerase III (C). This single copy gene was located near the centromere of chromosome XIV. It included a coding sequence of 317 amino acids that strictly matched two internal oligopeptides of C34. This polypeptide is a specific component of RNA polymerase III, with no significant homology to any other RNA polymerase subunit known so far. It is an essential subunit, since inactivation by deletion or nonsense mutations led to a recessive lethal phenotype. Moreover, a partially blocked mutant, rpc34-F297, had a reduced tRNA synthesis in vivo but no detectable effect on 5 S RNA synthesis. The latter phenotype was observed for all conditionally defective RNA polymerase III mutants isolated so far.

The conserved amino-terminal region of the largest subunit of yeast RNA polymerase C is capable of binding zinc ions in vitro. By oligonucleotide-directed mutagenesis, we show that the putative zinc-binding motif CX2CX6-12CXGHXGX24-37CX2C, present in the largest subunit of all eukaryotic and archaebacterial RNA polymerases, is essential for the function of RNA polymerase C. All mutations in the invariant cysteine and histidine residues conferred a lethal phenotype. We also obtained two conditional thermosensitive mutants affecting this region. One of these produced a form of RNA polymerase C which was thermosensitive and unstable in vitro. This instability was correlated with the loss of three of the subunits which are specific to RNA polymerase C: C82, C34, and C31.

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.

Two-dimensional crystals of yeast RNA polymerase A (I) were obtained by interaction with positively charged lipid layers. The analysis of single molecular images of lipid-bound RNA polymerases showed that the enzyme was preferentially oriented by the lipid phase, which probably facilitated crystallization. Electron micrographs of the crystals revealed a rectangular unit cell 25.8 nm by 45.6 nm in size containing four RNA polymerase dimers related by P22(1)2(1) symmetry. The projection map showed, at about 2.5 nm resolution, two different views of the enzyme characterized by two bent arms, which appeared to cross at one end. These arms are likely to contain the A190 and A135 subunits and delimit a 3 to 4 nm wide groove. Additional structural features were observed and compared to the Escherichia coli enzyme.

We have used gel retardation and DNase protection assays to investigate the trans-acting factors involved in the regulation of yeast RNA polymerase genes RPC160 and RPC40. The same binding component was found to interact with the promoter of the two genes, at a short distance (100-150 base pairs) from the transcription start sites. From its size, its DNA-binding specificity and its immunological properties, this factor appears to correspond to the autonomous replication sequences and silencer-binding factor ABF1/SBF-B. The interaction of ABF1 with the polymerase upstream box sequence was characterized using gel DNA-binding assay. The factor binds with high affinity to the polymerase upstream box sequence (Kapp = 5.10(-10) M). A mutational analysis showed that nine base pairs belonging to two separated attachment sites are involved in factor binding. The consensus sequence RTCRYB(N)4ACG was derived from the present binding studies. These data provide an experimental basis for evaluating the efficiency of known or potential ABF1 sites and for comparing several factors with ABF1-like binding properties.

Yeast RNA polymerase B (II) was incubated with a collection of 13 different nucleotide derivatives and affinity labeled by allowing DNA-directed phosphodiester bond formation. The 32P-labeled site was localized in the C-terminal part of the B150 subunit by microsequencing a proteolytic fragment, then further mapped by a combination of extensive or single-hit chemical cleavage reactions and analysis of the labeled peptide patterns. The affinity label was mapped to between Asn946 and Met999, within one of the nine regions that are conserved between B150 and the bacterial beta subunit. The results underscore the conservative evolution of the catalytic center of eukaryotic and bacterial RNA polymerases.

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

The structural features of the yeast DNA-dependent RNA polymerase A (I) were examined by Scanning Transmission Electron Microscopy. The enzyme was adsorbed in its monomeric form and negatively stained prior to digital image acquisition at low dose. The signal to noise ratio of single particle images was improved through averaging of a large number of previously aligned and partitioned images. Six classes of images were obtained reproducibly which corresponded to different projections of the enzyme. The enzyme structure was characterized by its elongated shape of 15.5 by 10.5 nm and by the presence of two curved arms which defined a longitudinal cleft. By analogy with theEscherichia coli enzyme, these arms could correspond to the two large subunits A135 and A190.

We report the identification and purification of a yeast factor functionally homologous to the human upstream element factor (UEFh). Although the yeast protein (UEFy) has a higher molecular weight than the HeLa UEF (60 kD versus 45 kD) both have identical DNA-binding properties: the purified UEFy recognizes the Adenovirus 2 (Ad2) major late promoter upstream element (MLP-UE; from nucleotide -49 to -67) as well as the IVa2 upstream element (IVa2-UE; from nucleotide -98 to -122) with a higher affinity for the MLP-UE than for the IVa2-UE. Based on its DNA binding specificity, size and thermostability, the UEFy protein appears also similar or equivalent to the centromere binding protein CP1. In a competition assay with oligonucleotides containing the MLP-UE binding site, a drastic reduction of Ad2 MLP transcription was observed both in a HeLa and in a yeast cell free system, which was restored by addition of either the purified UEFh or UEFy proteins. We conclude that both UEFh and UEFy activate transcription from the Ad2 MLP upon binding to the upstream element, whatever is the in vitro cell-free system (yeast or HeLa). This indicate that some regulatory function represented by the upstream element and its cognate factor, is well conserved between human and yeast.

We present a reproducible method for the preparation of nuclear extracts from the yeast Saccharomyces cerevisiae that support efficient RNA polymerase B (II)-dependent transcription. Extracts from both a crude nuclear fraction and Percoll-purified nuclei are highly active for site-specific initiation and transcription of a G-free cassette under the Adenovirus major late promoter. At optimal extract concentrations transcription is at least 5 times more efficient with the yeast extracts than with HeLa whole cell extracts. We show that the transcriptional activity is sensitive to alpha-amanitin and to depletion of factor(s) recognizing the TATA-box of the promoter. The in vitro reaction showed maximal activity after 45 min, was very sensitive to Cl-, but was not affected by high concentrations of potassium. We find that the efficiency of in vitro transcription in nuclear extracts is reproducibly high when spheroplasting is performed with a partially purified beta 1,3-glucanase (lyticase). Therefore a simplified method to isolate the lyticase from the supernatant of Oerskovia xanthineolytica is also presented.

International audience; Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, τA and τB, using the baculovirus expression system. The B block-binding module, rτB, was reconstituted with rτ138, rτ91, and rτ60 subunits. rτ131, rτ95, and rτ55 formed also a stable complex, rτA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B″ fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

The transcription factor IIIC (TFIIIC) is a multisubunit DNA-binding factor required for promoter recognition and TFIIIB assembly on tRNA genes transcribed by RNA polymerase III. Yeast TFIIIC consists of six subunits, organized in the two globular subcomplexes tauA and tauB, which recognize two internal tDNA promoter elements, the A and the B block, respectively. As a first step toward a detailed structural analysis of TFIIIC, we report here the expression, proteolytic analysis, reconstitution, and crystallization of the complex between yeast TFIIIC subunits tau91 and tau60. Proteolysis provided an insight into the domain structure of tau60 and tau91. Both the proteins form a stable complex that does not require an N-terminal, protease-sensitive extension of tau91. Crystals diffracting beyond 3.2 A were obtained from a complex formed by full-length tau60 and the N-terminally truncated form of tau91 lacking this extension.

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.

The yeast transcription factor IIIC (TFIIIC) is organized in two distinct multisubunit domains, tauA and tauB, that are respectively responsible for TFIIIB assembly and stable anchoring of TFIIIC on the B block of tRNA genes. Surprisingly, we found that the removal of tauA by mild proteolysis stabilizes the residual tauB.DNA complexes at high temperatures. Focusing on the well conserved tau95 subunit that belongs to the tauA domain, we found that the tau95-E447K mutation has long distance effects on the stability of TFIIIC.DNA complexes and start site selection. Mutant TFIIIC.DNA complexes presented a shift in their 5' border, generated slow-migrating TFIIIB.DNA complexes upon stripping TFIIIC by heparin or heat treatment, and allowed initiation at downstream sites. In addition, mutant TFIIIC.DNA complexes were highly unstable at high temperatures. Coimmunoprecipitation experiments indicated that tau95 participates in the interconnection of tauA with tauB via its contacts with tau138 and tau91 polypeptides. The results suggest that tau95 serves as a scaffold critical for tauA.DNA spatial configuration and tauB.DNA stability.

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.