Mutations of RNA polymerase II activate key genes of the nucleoside triphosphate biosynthetic pathways.

The yeast URA2 gene, encoding the rate-limiting enzyme of UTP biosynthesis, is transcriptionally activated by UTP shortage. In contrast to other genes of the UTP pathway, this activation is not governed by the Ppr1 activator. Moreover, it is not due to an increased recruitment of RNA polymerase II at the URA2 promoter, but to its much more effective progression beyond the URA2 mRNA start site(s). Regulatory mutants constitutively expressing URA2 resulted from cis-acting deletions upstream of the transcription initiator region, or from amino-acid replacements altering the RNA polymerase II Switch 1 loop domain, such as rpb1-L1397S. These two mutation classes allowed RNA polymerase to progress downstream of the URA2 mRNA start site(s). rpb1-L1397S had similar effects on IMD2 (IMP dehydrogenase) and URA8 (CTP synthase), and thus specifically activated the rate-limiting steps of UTP, GTP and CTP biosynthesis. These data suggest that the Switch 1 loop of RNA polymerase II, located at the downstream end of the transcription bubble, may operate as a specific sensor of the nucleoside triphosphates available for transcription.

Non-canonical DNA transcription enzymes and the conservation of two-barrel RNA polymerases.

DNA transcription depends on multimeric RNA polymerases that are exceptionally conserved in all cellular organisms, with an active site region of >500 amino acids mainly harboured by their Rpb1 and Rpb2 subunits. Together with the distantly related eukaryotic RNA-dependent polymerases involved in gene silencing, they form a monophyletic family of ribonucleotide polymerases with a similarly organized active site region based on two double-Psi barrels. Recent viral and phage genome sequencing have added a surprising variety of putative nucleotide polymerases to this protein family. These proteins have highly divergent subunit composition and amino acid sequences, but always contain eight invariant amino acids forming a universally conserved catalytic site shared by all members of the two-barrel protein family. Moreover, the highly conserved 'funnel' and 'switch 2' components of the active site region are shared by all putative DNA-dependent RNA polymerases and may thus determine their capacity to transcribe double-stranded DNA templates.

Early evolution of eukaryotic DNA-dependent RNA polymerases.

Eukaryotic DNA-dependent RNA polymerases (Pol I-III) share a conserved core of 12 subunits, which is closely related to archaeal RNA polymerases. Rpb8, a subunit found in Pol I, II and III, was thought to be restricted to eukaryotes. We show here that Rpb8 closely resembles an archaeal protein called G, found only in Crenarchaea, which identifies a last missing link between the core structure of archaeal and eukaryotic RNA polymerases.

Crucial role of a dicarboxylic motif in the catalytic center of yeast RNA polymerases.

The catalytic center of yeast RNA polymerase II and III contains an acidic loop borne by their second largest subunit (Rpb2-(832)GYNQED(837), Rpc128-(764)GYDIED(769)) and highly conserved in all cellular and viral DNA-dependent RNA polymerases. A site-directed mutagenesis of this dicarboxylic motif reveals its strictly essential character in RNA polymerase III, with a slightly less stringent pattern in RNA polymerase II, where rpb2-E836Q and other substitutions completely prevent growth, whereas rpb2-E836A combines a dominant growth defect with severe lethal sectoring. A mild but systematic increase in RNA polymerase occupancy and a strict dependency on the transcript cleavage factor TFIIS (Dst1) also suggest a slower rate of translocation or higher probability of transcriptional stalling in this mutation. A conserved nucleotide triphosphate funnel domain binds the Rpb2-(832)GYNQED(837) loop by an Rpb2-R(1020)/Rpb2-D(837) salt-bridge. Molecular dynamic simulations reveal a second bridge (Rpb1-K(752)/Rpb2-E(836)), which may account for the critical role of the invariant Rpb2-E(836). Rpb2-E(836) and the funnel domain are not found among the RNA-dependent eukaryotic RNA polymerases and may thus represent a specific adaptation to double-stranded DNA templates.

Functional organization of the Rpb5 subunit shared by the three yeast RNA polymerases.

Rpb5, a subunit shared by the three yeast RNA polymerases, combines a eukaryotic N-terminal module with a globular C-end conserved in all non-bacterial enzymes. Conditional and lethal mutants of the moderately conserved eukaryotic module showed that its large N-terminal helix and a short motif at the end of the module are critical in vivo. Lethal or conditional mutants of the C-terminal globe altered the binding of Rpb5 to Rpb1-beta25/26 (prolonging the Bridge helix) and Rpb1-alpha44/47 (ahead of the Switch 1 loop and binding Rpb5 in a two-hybrid assay). The large intervening segment of Rpb1 is held across the DNA Cleft by Rpb9, consistent with the synergy observed for rpb5 mutants and rpb9Delta or its RNA polymerase I rpa12Delta counterpart. Rpb1-beta25/26, Rpb1-alpha44/45 and the Switch 1 loop were only found in Rpb5-containing polymerases, but the Bridge and Rpb1-alpha46/47 helix bundle were universally conserved. We conclude that the main function of the dual Rpb5-Rpb1 binding and the Rpb9-Rpb1 interaction is to hold the Bridge helix, the Rpb1-alpha44/47 helix bundle and the Switch 1 loop into a closely packed DNA-binding fold around the transcription bubble, in an organization shared by the two other nuclear RNA polymerases and by the archaeal and viral enzymes.

Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III.

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner.

An Rpb4/Rpb7-like complex in yeast RNA polymerase III contains the orthologue of mammalian CGRP-RCP.

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.

Rpa43 and its partners in the yeast RNA polymerase I transcription complex.

An Rpa43/Rpa14 stalk protrudes from RNA polymerase I (RNAPI), with homology to Rpb7/Rpb4 (RNAPII), Rpc25/Rpc17 (RNAPIII) and RpoE/RpoF (archaea). In fungi and vertebrates, Rpa43 contains hydrophilic domains forming about half of its size, but these domains lack in Schizosaccharomyces pombe and most other eukaryote lineages. In Saccharomyces cerevisiae, they can be lost with little or no growth effect, as shown by deletion mapping and by domain swapping with fission yeast, but genetically interact with rpa12Δ, rpa34Δ or rpa49Δ, lacking non-essential subunits important for transcript elongation. Two-hybrid data and other genetic evidence suggest that Rpa43 directly bind Spt5, an RNAPI elongation factor also acting in RNAPII-dependent transcription, and may also interact with the nucleosomal chaperone Spt6.

Structure-function analysis of RNA polymerases I and III.

Recent advances in elucidating the structure of yeast Pol I and III are based on a combination of X-ray crystal analysis, electron microscopy and homology modelling. They allow a better comparison of the three eukaryotic nuclear RNA polymerases, underscoring the most obvious difference existing between the three enzymes, which lies in the existence of additional Pol-I-specific and Pol-III-specific subunits. Their location on the cognate RNA polymerases is now fairly well known, suggesting precise hypotheses as to their function in transcription during initiation, elongation, termination and/or reinitiation. Unexpectedly, even though Pol I and III, but not Pol II, have an intrinsic RNA cleavage activity, it was found that TFIIS Pol II cleavage stimulation factor also played a general role in Pol III transcription.

Two RNA polymerase I subunits control the binding and release of Rrn3 during transcription.

Rpa34 and Rpa49 are nonessential subunits of RNA polymerase I, conserved in species from Saccharomyces cerevisiae and Schizosaccharomyces pombe to humans. Rpa34 bound an N-terminal region of Rpa49 in a two-hybrid assay and was lost from RNA polymerase in an rpa49 mutant lacking this Rpa34-binding domain, whereas rpa34Delta weakened the binding of Rpa49 to RNA polymerase. rpa34Delta mutants were caffeine sensitive, and the rpa34Delta mutation was lethal in a top1Delta mutant and in rpa14Delta, rpa135(L656P), and rpa135(D395N) RNA polymerase mutants. These defects were shared by rpa49Delta mutants, were suppressed by the overexpression of Rpa49, and thus, were presumably mediated by Rpa49 itself. rpa49 mutants lacking the Rpa34-binding domain behaved essentially like rpa34Delta mutants, but strains carrying rpa49Delta and rpa49-338::HIS3 (encoding a form of Rpa49 lacking the conserved C terminus) had reduced polymerase occupancy at 30 degrees C, failed to grow at 25 degrees C, and were sensitive to 6-azauracil and mycophenolate. Mycophenolate almost fully dissociated the mutant polymerase from its ribosomal DNA (rDNA) template. The rpa49Delta and rpa49-338::HIS3 mutations had a dual effect on the transcription initiation factor Rrn3 (TIF-IA). They partially impaired its recruitment to the rDNA promoter, an effect that was bypassed by an N-terminal deletion of the Rpa43 subunit encoded by rpa43-35,326, and they strongly reduced the release of the Rrn3 initiation factor during elongation. These data suggest a dual role of the Rpa49-Rpa34 dimer during the recruitment of Rrn3 and its subsequent dissociation from the elongating polymerase.

Genome-wide location analysis reveals a role of TFIIS in RNA polymerase III transcription.

TFIIS is a transcription elongation factor that stimulates transcript cleavage activity of arrested RNA polymerase II (Pol II). Recent studies revealed that TFIIS has also a role in Pol II transcription initiation. To improve our understanding of TFIIS function in vivo, we performed genome-wide location analysis of this factor. Under normal growth conditions, TFIIS was detected on Pol II-transcribed genes, and TFIIS occupancy was well correlated with that of Pol II, indicating that TFIIS recruitment is not restricted to NTP-depleted cells. Unexpectedly, TFIIS was also detected on almost all Pol III-transcribed genes. TFIIS and Pol III occupancies correlated well genome-wide on this novel class of targets. In vivo, some dst1 mutants were partly defective in tRNA synthesis and showed a reduced Pol III occupancy at the restrictive temperature. In vitro transcription assays suggested that TFIIS may affect Pol III start site selection. These data provide strong in vivo and in vitro evidence in favor of a role of TFIIS as a general Pol III transcription factor.

Rpc25, a conserved RNA polymerase III subunit, is critical for transcription initiation.

Rpc25 is a strongly conserved subunit of RNA polymerase III with homology to Rpa43 in RNA polymerase I, Rpb7 in RNA polymerase II and the archaeal RpoE subunit. A central domain of Rpc25 can replaced the corresponding region of Rpb7 with little or no growth defect, underscoring the functional relatedness of these proteins. Rpc25 forms a heterodimer with Rpc17, another conserved component of RNA polymerase III. A conditional mutant (rpc25-S100P) impairs this interaction. rpc25-S100P and another conditional mutant obtained by complementation with the Schizosaccharomyces pombe subunit (rpc25-Sp) were investigated for the properties of their purified RNA polymerase III. The mutant enzymes were defective in the specific synthesis of pre-tRNA transcripts but acted at a wild-type level on poly[d(A-T)] templates. They were also indistinguishable from wild type in transcript elongation, cleavage and termination. These data indicate that Rpc25 is needed for transcription initiation but is not critical for the elongating properties of RNA polymerase III.

Hmo1, an HMG-box protein, belongs to the yeast ribosomal DNA transcription system.

Hmo1 is one of seven HMG-box proteins of Saccharo myces cerevisiae. Null mutants have a limited effect on growth. Hmo1 overexpression suppresses rpa49-Delta mutants lacking Rpa49, a non-essential but conserved subunit of RNA polymerase I corresponding to the animal RNA polymerase I factor PAF53. This overexpression strongly increases de novo rRNA synthesis. rpa49-Delta hmo1-Delta double mutants are lethal, and this lethality is bypassed when RNA polymerase II synthesizes rRNA. Hmo1 co-localizes with Fob1, a known rDNA-binding protein, defining a narrow territory adjacent to the nucleoplasm that could delineate the rDNA nucleolar domain. These data identify Hmo1 as a genuine RNA polymerase I factor acting synergistically with Rpa49. As an HMG-box protein, Hmo1 is remotely related to animal UBF factors. hmo1-Delta and rpa49-Delta are lethal with top3-Delta DNA topoisomerase (type I) mutants and are suppressed in mutants lacking the Sgs1 DNA helicase. They are not affected by top1-Delta defective in Top1, the other eukaryotic type I topoisomerase. Conversely, rpa34-Delta mutants lacking Rpa34, a non-essential subunit associated with Rpa49, are lethal in top1-Delta but not in top3-Delta.

Rpa12p, a conserved RNA polymerase I subunit with two functional domains.

Rpa12p is a subunit of RNA polymerase I formed of two zinc-binding domains. The N-terminal zinc region (positions 1-60) is poorly conserved from yeast to man. The C-terminal domain contains an invariant Q.RSADE.T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N-terminal domain fail to grow at 34 degrees C, are sensitive to nucleotide-depleting drugs and become lethal in rpa14-Delta mutants lacking the non-essential RNA polymerase I subunit Rpa14p. They also strongly alter the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific two-hybrid interaction with the second largest subunit. In all these respects, N-terminal deletions behave like full deletions. In contrast, C-terminal deletions retaining only the first N-terminal 60 amino acids are indistinguishable from wild type. Thus, the N-terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I and is the only critical part of that subunit in vivo.

Members of the SAGA and Mediator complexes are partners of the transcription elongation factor TFIIS.

TFIIS, an elongation factor encoded by DST1 in Saccharomyces cerevisiae, stimulates transcript cleavage in arrested RNA polymerase II. Two components of the RNA polymerase II machinery, Med13 (Srb9) and Spt8, were isolated as two-hybrid partners of the conserved TFIIS N-terminal domain. They belong to the Cdk8 module of the Mediator and to a subform of the SAGA co-activator, respectively. Co-immunoprecipitation experiments showed that TFIIS can bind the Cdk8 module and SAGA in cell-free extracts. spt8Delta and dst1Delta mutants were sensitive to nucleotide-depleting drugs and epistatic to null mutants of the RNA polymerase II subunit Rpb9, suggesting that their elongation defects are mediated by Rpb9. rpb9Delta, spt8Delta and dst1Delta were lethal in cells lacking the Rpb4 subunit. The TFIIS N-terminal domain is also strictly required for viability in rpb4Delta, although it is not needed for binding to RNA polymerase II or for transcript cleavage. It is proposed that TFIIS and the Spt8-containing form of SAGA co-operate to rescue RNA polymerase II from unproductive elongation complexes, and that the Cdk8 module temporarily blocks transcription during transcript cleavage.

The Rpb9 subunit of RNA polymerase II binds transcription factor TFIIE and interferes with the SAGA and elongator histone acetyltransferases.

Rpb9 is a small subunit of yeast RNA polymerase II participating in elongation and formed of two conserved zinc domains. rpb9 mutants are viable, with a strong sensitivity to nucleotide-depleting drugs. Deleting the C-terminal domain down to the first 57 amino acids has no detectable growth defect. Thus, the critical part of Rpb9 is limited to a N-terminal half that contacts the lobe of the second largest subunit (Rpb2) and forms a beta-addition motif with the "jaw" of the largest subunit (Rpb1). Rpb9 has homology to the TFIIS elongation factor, but mutants inactivated for both proteins are indistinguishable from rpb9 single mutants. In contrast, rpb9 mutants are lethal in cells lacking the histone acetyltransferase activity of the RNA polymerase II Elongator and SAGA factors. In a two-hybrid test, Rpb9 physically interacts with Tfa1, the largest subunit of TFIIE. The interacting fragment, comprising amino acids 62-164 of Tfa1, belongs to a conserved zinc motif. Tfa1 is immunoprecipitated by RNA polymerase II. This co-purification is strongly reduced in rpb9-Delta, suggesting that Rpb9 contributes to the recruitment of TFIIE on RNA polymerase II.