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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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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 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.

Rpb4p and Rpb7p are two subunits of the yeast RNA polymerase II, which form a subcomplex that can dissociate from the enzyme in vitro. Whereas RPB7 is essential, RPB4 is dispensable for cellular viability. However, the rpb4 null mutant is heat-sensitive, and it has been suggested that Rpb4p is an essential component for cellular stress response. To examine this hypothesis, we used two-dimensional gel electrophoresis to analyze the protein expression pattern of the rpb4 null mutant in response to heat shock, oxidative stress, osmotic stress, and in the post-diauxic phase. We show that this mutant is not impaired in stress induced transcriptional activation: the absence of heat shock response of the mutant is due to a general defect in RNA polymerase II activity at high temperature. Under this condition, Rpb4p is necessary to maintain the polymerase activity in vivo. The heat growth defect of the rpb4 null mutant can be partially suppressed by overexpression of RPB7, suggesting that Rpb4p maintains or stabilizes Rpb7p in the RNA polymerase. We also demonstrate that rpb4 null mutant is an appropriate tool to analyze the involvement of transcriptional events in the survival and adaptation to heat shock or other stresses.

The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein–protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10α, and ABC10β), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

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.

Two-dimensional crystals of yeast RNA polymerase I dimers were obtained upon interaction with positively charged lipid layers. A three-dimensional surface model of the enzyme was determined by analyzing tilted crystalline areas and by taking advantage of the non-crystallographic internal symmetry of the dimer to correct for the missing viewing directions. The structure shows, at approximately 3 nm resolution, an irregularly shaped molecule 11 nm x 11 nm x 15 nm in size characterized by a 3 nm wide and 10 nm long groove which constitutes a putative DNA binding site. The overall structure is similar to the Escherichia coli holo enzyme and the yeast RNA polymerase II delta 4/7 structures. The most remarkable structural feature is a finger-shaped stalk which partially occludes the entrance of the groove and forms a 2.5 nm wide channel. We discuss the possible location of the catalytic centre and of the carboxy-terminal region of the beta-like subunit in the channel. The interference of different DNA fragments with RNA polymerase dimerization and crystallization indicates the orientation of the template in the putative DNA binding groove.

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.

RPC53 has previously been shown to encode an essential subunit required for tRNA gene transcription by RNA polymerase C in vivo (Mann, C., Micouin, J.-Y., Chiannilkulchai, N., Treich, I., Buhler, J.-M., and Sentenac, A. (1992) Mol. Cell. Biol. 12, in press). In this paper, we have determined that an unusual rho+ lethality associated with the rpc53::HIS3-1 disruption mutation is due to the inadvertent formation of a Pet56-C53 fusion protein. This fusion protein is missorted to mitochondria, thereby reducing the quantity of the C53 subunit available for RNA polymerase C assembly. We show that the carboxyl-terminal region of C53 contains the essential functional domain of the subunit and that a mutant RNA polymerase containing only this domain is thermolabile for its function in vivo and in vitro. The thermolability of the carboxyl-terminal C53 domain is suppressed by five different genes on multicopy plasmids, including RPC160, encoding the largest subunit of RNA polymerase C and SSD1/SRK1, which has been implicated in the activity of protein phosphatases.

In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1). In both cases, the labeled site was localized to the C-terminal part of the B150 subunit. The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999. In the present work, we have mutagenized to arginine the five lysines present in domain H. Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled. Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent. These two lysines were each found to be essential for cell viability. Derivative VIII labeled another domain in addition to domain H. Supported by analogous results obtained for E. coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I. Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII. These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site.

Yeast nuclear RNA polymerases are multisubunit enzymes that contain in common some small subunits. We show that the smallest, a 10-kDa component of three enzymes (A10, B10, and C10), is heterogeneous. In each case, it can be resolved into two distinct polypeptides (alpha and beta) by reverse-phase chromatography. A10 alpha, B10 alpha, and C10 alpha were indistinguishable on the basis of their electrophoretic and chromatographic behaviors, characteristic silver staining, and tryptic peptide analysis. All three polypeptides are blocked at their amino termini. By the same criteria, A10 beta, B10 beta, and C10 beta were also indistinguishable. The amino-terminal sequence of A10 beta and C10 beta corresponded to that of subunit B10 recently cloned by Woychik and Young (Woychik, N. A., and Young, R. A. (1990) J. Biol. Chem. 265, 17816-17819). Thus, the three forms of RNA polymerase share two additional and distinct polypeptides, ABC10 alpha and ABC10 beta, that therefore can be considered bona fide subunits of these enzymes. Interestingly, these two subunits bind zinc.

The zinc-binding subunits of yeast RNA polymerase A(I) and B(II) have been identified by a zinc-blotting technique. The two largest subunits of each enzyme (A190, A135, B220, and B150), as well as A12.2, A10, B44.5, B12.6, and B10, bind 65Zn(II). Predicted zinc-binding motifs have been noted in the NH2-terminal part of B220 and the COOH-terminal region of B150 subunits. Subdomains encompassing these motifs have been overproduced as MalE-fusion proteins and shown to retain zinc binding activity. Site-directed mutagenesis in the predicted metal-binding domain of B150 demonstrated its role in zinc binding. Mutations of cysteine residues C1163, C1166, C1182, and C1185 affected 65Zn2+ binding in vitro and caused a lethal or thermosensitive phenotype for growth. The ability to bind zinc is not sufficient for function since mutations in vicinal residues not affecting zinc binding were either lethal or thermosensitive. The role of zinc in RNA polymerase structure and function is discussed in the light of the present results.

Transcription of small genes by RNA polymerase III or C (pol III) involves many of the strategies that are used for transcription complex formation and occasionally the same components as those used by RNA polymerase II or B (pol II). Transcription complex formation is a multistep process that leads to the binding of a single initiation factor, TFIIIB, which in turn directs the selection of pol III. The general transcription factor TFIID can be involved in both pol II and pol III transcription. These and other similarities point towards a unifying mechanism for eukaryotic transcription initiation.

The c-Myb protein is a sequence-specific DNA binding protein that activates transcription in hematopoietic cells. Three imperfect repeats (R1, R2, and R3) that contain regularly spaced tryptophan residues form the DNA binding domain of c-Myb. A fragment of c-Myb that contained the R2 and R3 regions bound specifically to a DNA sequence recognized by c-Myb plus ten additional base pairs at the 3' end of the element. The R2R3 fragment was predicted to contain two consecutive helix-turn-helix (HTH) motifs with unconventional turns. Mutagenesis of amino acids in R2R3 at positions that correspond to DNA-contacting amino acids in other HTH-containing proteins abolished specific DNA binding without affecting nonspecific DNA interactions.

Yeast RNA polymerases A (I) and C (III) share a subunit called AC19. The gene encoding AC19 has been isolated from yeast genomic DNA using oligonucleotide probes deduced from peptide sequences of the isolated subunit. This gene (RPC19) contains an intron-free open reading frame of 143 amino acid residues. RPC19 is a single copy gene that maps on chromosome II and is essential for cell viability. The amino acid sequence contains a sequence motif common to the Escherichia coli RNA polymerase alpha subunit, the Saccharomyces cerevisiae AC40 and B44.5 subunits, the human hRPB33 product, and the CnjC conjugation-specific gene product of Tetrahymena. The 5'-upstream region contains a sequence element, the PAC box, that has been conserved in at least 10 genes encoding subunits of RNA polymerases A and C.

Yeast RNA polymerase I contains 14 distinct polypeptides, including A43, a component of about 43 kDa. The corresponding gene, RPA43, encodes a 326-amino acid polypeptide matching the peptidic sequence of two tryptic fragments isolated from A43. Gene inactivation leads to a lethal phenotype that is rescued by a plasmid containing the 35S ribosomal RNA gene fused to the GAL7 promoter, which allows the synthesis of 35S rRNA by RNA polymerase II in the presence of galactose. A screening for mutants rescued by the presence of GAL7-35SrDNA identified a nonsense rpa43 allele truncating the protein at amino acid position 217. [3H]Uridine pulse labeling showed that this mutation abolishes 35S rRNA synthesis without significant effects on the synthesis of 5 S RNA and tRNAs. These properties establish that A43 is an essential component of RNA polymerase I. This highly hydrophilic phosphoprotein has a strongly acidic carboxyl-terminal domain, and shows no homology to entries in current sequence data banks, including all the genetically identified components of the other two yeast RNA polymerases. RPA43 mapped next to RPA190, encoding the largest subunit of polymerase I. These genes are divergently transcribed and may thus share upstream regulatory elements ensuring their co-regulation.

This review will attempt to cover the present information on the multiple forms of eukaryotic DNA-dependent RNA polymerases, both at the structural and functional level. Nuclear RNA polymerases constitute a group of three large multimeric enzymes, each with a different and complex subunit structure and distinct specificity. The review will include a detailed description of their molecular structure. The current approaches to elucidate subunit function via chemical modification, phosphorylation, enzyme reconstitution, immunological studies, and mutant analysis will be described. In vitro reconstituted systems are available for the accurate transcription of cloned genes coding for rRNA, tRNA, 5 SRNA, and mRNA. These systems will be described with special attention to the cellular factors required for specific transcription. A section on future prospects will address questions concerning the significance of the complex subunit structure of the nuclear enzymes; the organization and regulation of the gene coding for RNA polymerase subunits; the obtention of mutants affected at the level of factors, or RNA polymerases; the mechanism of template recognition by factors and RNA polymerase.

The subunits of yeast RNA polymerases A(I) and B(II) were characterized using several techniques. The present studies demonstrate that the A and B enzymes possess three subunits, which are indistinguishable on the basis of molecular weight, isoelectric point, and fingerprint pattern. The three common subunits belong to the small molecular weight components of the enzymes. By polyacrylamide gel electrophoresis with sodium dodecyl sulfate they migrate with apparent molecular weights of 27,000, 23,000, and 14,500, respectively. A two-dimensional subunit mapping technique on polyacrylamide gel was used to separate the subunits according to isoelectric point and molecular weight. The common polypeptides co-migrated on three spots corresponding to isoelectric points of 9.2 (27,000), 4.5 (23,000), and 4.6 (14,500). The fingerprints of the 35S-labeled tryptic peptides of the presumptive common subunits were found to be essentially identical. Finally, the presence of common subunits was supported by the fact that antibodies against pure RNA polymerase A cross-react with and inhibit RNA polymerase B. Except for the common subunits, it is likely that RNA polymerases A and B are primarily made of distinct gene products for the following reasons. A total of 13 polypeptide chains are present in enzyme A, whereas 10 polypeptides are found in enzyme B. The molecular weight, isoelectric point, and sulfur content of the majority of these polypeptide chains are different in the two enzymes. No similarity was found in the 35S-peptide fingerprint from a number of A and B subunits of slightly different molecular weight. Finally, antibodies against the largest subunit from RNA polymerase A do not cross-react with or inhibit RNA polymerase B. The data are discussed in terms of structural organization of eukaryotic RNA polymerases.

A method was devised for directing RNA polymerase on a single promoter site on T7 DNA. Initiation complexes were formed on each of the three main promoter sites using one dinucleotide plus one nucleoside triphosphate. The ternary initiation complexes are resistant to rifampicin action, to inhibition by (rI)n at 0 degrees C and are stable at high salt concentrations. A minimum of a trinucleotide is required to form a stable ternary complex. To determine which promoter site was selected by RNA polymerase during initiation, the (rI)n-resistant RNA was digested by RNAse III to generate three characteristic initiator RNA fragments, resolved by gel electrophoresis. The three major promoter sites could be selected individually by using different primer and substrate combinations ApC plus ATP selected promoter A3, CpG plus CTP selected A2 and CpC plus ATP specified preferentially A1. A number of primer-substrate combinations specified each site at low salt concentration but the substrate requirement became very stringent at high salt concentration, suggesting that the postulated local opening of the promoter site could be more or less extensive, depending on the ionic strength. The minimum opening observed at high salt concentration corresponded to the insertion of a leader trinucleotide sequence. The promoter region melted by RNA polymerase at low salt concentration was (G plus C)-rich and corresponded to about 9 to 11 base pairs. Sequences of the melting recognition regions were tentatively inferred from the results.

Antisera were raised against native RNA polymerases A or B, as well as against each individual subunit of RNA polymerase A from the yeast Saccharmoyces cerevisiae. The affinity spectrum of antibodies was evaluated by reacting electrophoretically separated enzyme subunits, transferred to a membrane, with 125I-labeled immunoglobulins. Alternatively, the subunit . immunoglobulin complex was revealed by 125I-labeled Protein A. Antibodies directed against native RNA polymerase A recognized the majority of the polypeptides forming the enzyme. When challenged with RNA polymerases B or C, this antibody preparation demonstrated the presence of polypeptides common to the three enzymes. A small cross-reaction was also found at the level of the large subunits of Enzyme B as well as some additional polypeptides of Enzyme C. Similar experiments with antibodies directed against native RNA polymerase B confirmed the presence of common subunits and also showed that the large polypeptides of the three enzymes share a few immunological determinants. Common subunits are AC40, ABC27, ABC23, AC19, and ABC14.5. Immunologically related sites were conserved in the large subunits of RNA polymerase A from remote yeast species. Similarly, yeast and wheat germ RNA polymerase B share immunological determinants on the large subunit as well as on a small peptide. On the other hand, there was no significant cross-reaction between yeast and mammalian Enzyme B or Escherichia coli RNA polymerase. Antibodies raised against the different polypeptide components of RNA polymerase A reacted specifically with the corresponding subunits. Inhibition studies with these subunit-specific antibodies showed that the common subunits are not always similarly exposed to antibody attack within the three enzymes. The data are discussed in terms of the structural similarity, organization and evolution of eukaryotic RNA polymerases.

A new RNA-dependent ATPase has been isolated from yeast chromatin extracts and partially characterized. The protein has a sedimentation coefficient of about 7 S. The enzyme hydrolyzes specifically ATP (or dATP) to ADP (or dADP) and Pi in the presence of Mg2+ or Mn2+ ions and requires a single-stranded polynucleotide as cofactor. The order of efficiency of synthetic polymers is poly(rU) > poly(rI) greater than or equal to poly(dU) > poly(rA) greater than or equal to poly(rC). Among natural polymers, single-stranded DNA and poly(rA)-containing mRNA from yeast are also active but less so than poly(rU). The enzyme exhibits a pH optimum of 8 and is fully inhibited by 0.25 M NaCl. The Km for ATP is0.2 mM. The resemblance between this ATPase and DNA-dependent ATPases from other sources, as well as the termination factor rho, is discussed.

The pattern of restriction DNA fragments transferred from agarose gel to nitrocellulose membranes (Southern's technique) can be affected by the salt concentration of the transfer solvent.

Yeast RNA polymerase A (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) can be converted to a new form of enzyme, called RNA polymerase A*, which is lacking two polypeptide chains of 48,000 and 37,000 daltons. Apart from these two missing polypeptides the subunit structures of RNA polymerases A and A* are indistinguishable. RNA polymerase A* differs from the complete enzyme in its electrophoretic and chromatographic behavior, template requirements, and alpha-amanitin sensitivity. RNA polymerase A* transcribes the alternated copolymer d(A-T)n with the same efficiency as RNA polymerase A but its specific activity is greatly reduced with native calf thymus DNA as template. The transcription of a variety of synthetic templates is also altered by removal of the two polypeptide chains. RNA polymerase A* is inhibited by high concentrations of alpha-amanitin (500 mug/ml), whereas RNA polymerase A is comparatively less sensitive to the toxic peptide. The data are discussed in terms of possible roles of the two dissociable polypeptides.

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from the yeast Saccharomyces cerevisiae using a large-scale procedure. The three steps of purification used were batch adsorption on phosphocellulose, phosphocellulose chromatography and, as the last step, GDP-Sepharose or Biorex column chromatography. The protein is very basic (pI = 9.2) and has an apparent molecular mass of 49 kDa, as determined by polyacrylamide gel electrophoresis using denaturing conditions. It is one of the most abundant proteins in yeast (about 5% of total soluble protein), as shown by two-dimensional gel electrophoresis and by immunological titration. A strong immunological and structural homology was found between yeast EF-1 alpha and elongation factors from other sources. Common immunological features were found between yeast and wheat germ EF-1 alpha. Tryptic hydrolysis of yeast EF-1 alpha in the presence of 25% glycerol generated a large trypsin-resistant polypeptide (Mr = 43,000) which had the same NH2-terminal sequence as the proteolyzed product from rabbit reticulocyte, Artemia salina EF-1 alpha and Escherichia coli EF-Tu. Completed DNA sequence determination of one structural gene for yeast EF-1 alpha confirmed a remarkable conservation of several protein sequence domains in yeast and animal EF-1 alpha (Cottrelle, P., Thiele, D., Price, V., Memet, S., Micouin, J.Y., Marck, C., Buhler, J.M. Sentenac, A., and Fromageot, P. (1985) J. Biol. Chem. 260, 3090-3096).

Two ribonucleases H, which specifically degrade RNA in RNA-DNA hybrid structures, have been extensively purified from extracts of Saccharomyces corevisiae. Ribonuclease H1 can be obtained in high yield by a rapid purification procedure mainly involving ammonium sulfate fractionation and phosphocellulose chromatography. The enzyme preparation was shown to be homogeneous by acrylamide gel electrophoresis with sodium dodecylsulfate. Ribonuclease H2 was also obtained in an essentially homogeneous form by phosphocellulose and Amberlite chromatography. About 60 mg of ribonuclease H1 and 2 mg of ribonuclease H2 were obtained from 100 g of wet cells. Using T7 DNA · RNA, hybrids, the specific activity of ribonuclease H2 was approximately 10-fold higher than that of ribonuclease H1.

Lomofungin is a potent inhibitor of RNA synthesis in yeast. Studies on the mode of action of the inhibitor were carried out using yeast RNA polymerases A and B and bacterial RNA polymerase. In vitro inhibition of RNA synthesis is independent of the nature and concentration of the template used and of the nucleoside triphosphate concentration. The extent of inhibition is strongly dependent upon the nature and concentration of divalent cations used to simulate transcription. The three RNA polymerases were inhibited to the same extent in the presence of Mn2+ ions whereas little inhibition was observed with Mg2+ ions. Spectrophotometric studies reveal the formation of different complexes between lomofungin and divalent cations (Mn2+, Mg2+, or Zn2+) with the respective stoichiometries of 0.5, 1, and 2 divalent cations per molecule of lomofungin. The complexes formed depend upon the nature of the divalent cation involved. No direct interaction between lomofungin and DNA could be observed in the presence of divalent cations but evidence is presented that lomofungin interacts with yeast RNA polymerase A. Inhibition of RNA synthesis occurs at the level of both chain initiation and elongation.

The template specificity of yeast RNA polymerase B (or II) has been investigated using single-stranded homopolymers, helical homopolymers, and alternated copolymers. The enzyme exhibited a remarkable base sequence specificity for single-stranded polynucleotides; only those containing pyrimdine nucleotides were transcribed with (dC)n g (dT)n. Homopolymer pairs were transcribed asymmetrically, the pyrimidine-containing strand acting as template. The best synthetic template was the alternated d(I-C)n copolymer. The results signal a preference by the enzyme for initiating in pyrimidine-rich and (dC)-rich regions of the DNA. The structure of the RNA product was studied with ribonucleases of different specificity, RNases A and Ti, RNase H, and RNase III. It appeared that the dissociation of the RNA product from the template strand was determined by the relative stabilities of the helical structures involved. Net synthesis of RNA was favored by the release of the RNA chain from the template. Among polyribonucleotides, (rC)n was a very efficient template, turnip yellow mosaic virus RNA had some activity, but no other ribopolymer tested was found to have a significant activity. Competition experiments with polyribonucleotides indicated a different specificity with respect to the binding reaction, since (rG)n and (rU)n, which had no template activity, are much better inhibitors of transcription than (rC)n.

Yeast transcription factor τ interacts with the A and B blocks of the intragenic promoter of tRNA genes. The structure of τ was investigated by identifying the polypeptide chains specifically complexed to the tRNA3Glu gene. Highly purified factor, obtained by an improved purification procedure, contained several polypeptide chains, four of which (Mr = 145,000, 135,000, 100,000 and 65,000) comigrated with τ-DNA complex by polyacrylamide gel electrophoresis. Antibodies raised against the 145- and 100-kDa components altered the migration of τ-DNA complexes in band shift assays and inhibited tRNA synthesis in a reconstituted transcription system. These components are immunologically unrelated proteins. By UV cross-linking to 32P-body-labeled tDNA followed by extensive DNase treatment, two polypeptides of the same size (145 and 100 kDa) were found to be radioactively labeled. Factor τ, therefore, appears to be a multisubunit DNA-binding protein with two distinct polypeptides contributing to DNA recognition. Limited proteolysis of τ generated a protease-resistant τB (τB) domain that binds solely to the B block. τB-tDNA complexes were recognized by anti-145 IgG and contained a 120-kDa polypeptide that could originate from the 145-kDa component by proteolysis. These results strongly suggest that the 145-kDa polypeptide belongs to τB and is responsible for B block binding.

Summary Using an immunochemical approach homologies between single components of DNA-dependent RNA polymerases from eubacteria, archaebacteria and eukaryotes were investigated. The largest components of all RNA polymerases included in this study are homologous to one another indicating a monophyletic origin of these proteins. Immunological crossreactions show that one of the large subunits present in the enzymes of sulfur-dependent archaebacteria is split into two smaller components in methanogens and halophiles. One of these smaller components roughly corresponds to the second largest subunit of the three eukaryotic enzymes whereas the other one shares antigenic determinants with subunit s of eubacterial RNA polymerases. Semi-quantitative evaluation of the data suggests that the three nuclear RNA polymerases of eukaryotes have evolved from an ancestral enzyme of the type that is found in sulfur-dependent archaebacteria.

A new in vitro approach is used to investigate the specificity of purified yeast RNA polymerase B (II). The template is supercoiled, the transcription is primed by a dinucleotide, and the transcripts are analyzed by polyacrylamide gel electrophoresis after synthesis in the absence of one nucleoside triphosphate. Under these conditions, two recombinant plasmids carrying the gene or part of the gene for yeast alcohol dehydrogenase I direct the synthesis of a very limited number of oligonucleotides. Elongation of these prelabeled oligomers, using unlabeled substrates, occurs in a discontinuous way. A major transcript of 200 nucleotides accumulates transiently. Southern hybridization shows that it is initiated about 1,400 bases upstream from the origin of the yeast alcohol dehydrogenase I gene. A minor start was identified, by a modified runoff experiment, at position -35 from the AUG initiation codon. The location of this site is related to the presumptive in vivo transcription starts. The selectivity disappears when the template is a truncated DNA. Then, initiation occurs predominantly at nicks introduced by the restriction enzymes.