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RRN6 and RRN7 encode subunits of a multiprotein complex essential for the initiation of rDNA transcription by RNA polymerase I in Saccharomyces cerevisiae
Abstract and Figures
Previously, we have isolated mutants of Saccharomyces cerevisiae primarily defective in the transcription of 35S rRNA genes by RNA polymerase I and have identified a number of genes (RRN genes) involved in this process. We have now cloned the RRN6 and RRN7 genes, determined their nucleotide sequences, and found that they encode proteins of calculated molecular weights of 102,000 and 60,300, respectively. Extracts prepared from rrn6 and rrn7 mutants were defective in in vitro transcription of rDNA templates. We used extracts from strains containing epitope-tagged wild-type Rrn6 or Rrn7 proteins to purify protein components that could complement these mutant extracts. By use of immunoaffinity purification combined with biochemical fractionation, we obtained a highly purified preparation (Rrn6/7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparent molecular weight of 66,000, but which did not contain the TATA-binding protein (TBP). This complex complemented both rrn6 and rrn7 mutant extracts. Template commitment experiments carried out with this purified Rrn6/7 complex and with rrn6 mutant extracts have demonstrated that the Rrn6/7 complex does not bind stably to the rDNA template by itself, but its binding is dependent on the initial binding of some other factor(s) and that the Rrn6/7 complex is required for the formation of a transcription-competent preinitiation complex. These observations are discussed in comparison to in vitro rDNA transcription systems from higher eukaryotes.
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... The degradation of RNA polymerase I in response to starvation may be another mechanism for S. cerevisiae rRNA repression. RNA polymerase I, in concert with core factor CF [61], upstream activation factor UAF [62], TATA-binding protein TBP [63], and monomeric factor Rrn3 [64], are recruited to the rDNA promoter. Among them, rapamycin treatment diminishes RRN3 transcription and causes degradation of pre-existing Rrn3 [65]. ...
Restricting ribosome biosynthesis and assembly in response to nutrient starvation is a universal phenomenon that enables cells to survive with limited intracellular resources. When cells experience starvation, nutrient signaling pathways, such as the target of rapamycin (TOR) and protein kinase A (PKA), become quiescent, leading to several transcription factors and histone modification enzymes cooperatively and rapidly repressing ribosomal genes. Fission yeast has factors for heterochromatin formation similar to mammalian cells, such as H3K9 methyltransferase and HP1 protein, which are absent in budding yeast. However, limited studies on heterochromatinization in ribosomal genes have been conducted on fission yeast. Herein, we shed light on and compare the regulatory mechanisms of ribosomal gene transcription in two species with the latest insights.
... In general, the activity of cellular extracts on rDNA templates in vitro reflects the growth rate of the cells from which they were prepared (36,114,251). Despite the usefulness of the system, only recently has some progress been made in the dissection of the yeast PolI transcriptional apparatus in vitro (160,271). Schnapp et al. have identified TIF-IA as a protein that is, at least in part, responsible for the growth rate-dependent activity of cellular extracts in mice (287). TIF-IA is 75 kDa in size and is thought to be the mammalian homolog of the prokaryotic sigma factor (288). ...
The control of rRNA synthesis in response to both extra- and intracellular signals has been a subject of interest to microbial physiologists for nearly four decades, beginning with the observations that Salmonella typhimurium cells grown on rich medium are larger and contain more RNA than those grown on poor medium. This was followed shortly by the discovery of the stringent response in Escherichia coli, which has continued to be the organism of choice for the study of rRNA synthesis. In this review, we summarize four general areas of E. coli rRNA transcription control: stringent control, growth rate regulation, upstream activation, and anti-termination. We also cite similar mechanisms in other bacteria and eukaryotes. The separation of growth rate-dependent control of rRNA synthesis from stringent control continues to be a subject of controversy. One model holds that the nucleotide ppGpp is the key effector for both mechanisms, while another school holds that it is unlikely that ppGpp or any other single effector is solely responsible for growth rate-dependent control. Recent studies on activation of rRNA synthesis by cis-acting upstream sequences has led to the discovery of a new class of promoters that make contact with RNA polymerase at a third position, called the UP element, in addition to the well-known -10 and -35 regions. Lastly, clues as to the role of antitermination in rRNA operons have begun to appear. Transcription complexes modified at the antiterminator site appear to elongate faster and are resistant to the inhibitory effects of ppGpp during the stringent response.
... These "rrn" mutants identified genes encoding unique subunits of pol I (22,24), as well as genes encoding other components of the committed template. The development of an in vitro transcription system (25)(26)(27)(28)(29) led to the combination of genetic and biochemical approaches that enhanced our understanding of the mechanism of rDNA transcription in yeast. ...
Our knowledge of the mechanism of rDNA transcription has benefitted from the combined application of genetic and biochemical techniques in yeast. Nomura's laboratory developed a system in yeast to identify genes essential for ribosome biogenesis. Such systems have allowed investigators to determine if a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast. Thus, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an auxin-inducible degron that enables combining a "genetics-like"approach with biochemistry to study mammalian rDNA transcription. We now show that the mammalian orthologue of yeast RPA49, PAF53, is required for rDNA transcription and mitotic growth. We have studied the domains of the protein required for activity. We have found that the C-terminal, DNA-binding domain (tWH), the heterodimerization and the linker domain were essential. Analysis of the linker identified a putative helix-turn-helix (HTH) DNA-binding domain. This helix-turn-helix (HTH) constitutes a second DNA-binding domain within PAF53. The HTH of the yeast and mammalian orthologues is essential for function. In summary, we show that an auxin-dependent degron system can be used to rapidly deplete nucleolar proteins in mammalian cells, that PAF53 is necessary for rDNA transcription and cell growth, and that all three PAF53 domains are necessary for its function.
... These "rrn" mutants identified genes encoding unique subunits of pol I (22,24), as well as genes encoding other components of the committed template. The development of an in vitro transcription system (25)(26)(27)(28)(29) led to the combination of genetic and biochemical approaches that enhanced our understanding of the mechanism of rDNA transcription in yeast. ...
Our knowledge of the mechanism of rDNA transcription has benefitted from the combined application of genetic techniques in yeast, and progress on the biochemistry of the various components of yeast rDNA transcription. Nomura′s laboratory derived a system in yeast for screening for mutants essential for ribosome biogenesis. Such systems have allowed investigators to not only determine if a gene was essential, but to analyze domains of the proteins for different functions in rDNA transcription in vivo. However, because there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an inducible degron that allows us to combine a ′genetics-like′ approach to studying mammalian rDNA transcription with biochemistry. Using this system, we show that the mammalian homologue of yeast A49, PAF53, is required for rDNA transcription and mitotic growth. Further, we have been able to study the domains of the protein required for activity. We have found that while the C-terminal, DNA-binding domain (tWH) was necessary for complete function, the heterodimerization and linker domains were also essential. Analysis of the linker identified a putative DNA-binding domain. We have confirmed that the helix-turn-helix (HTH) of the linker constitutes a second DNA-binding domain within PAF53 and that the HTH is essential for PAF53 function.
... Both, mammalian and yeast promoters are arranged in two main domains including a core element (CE, Fig 1A), required for transcription initiation in vitro, and an upstream (control) element (UE in yeast (Fig 1A), UCE in mammals), supporting activated transcription under certain conditions (reviewed in [3]). Whereas promoter DNA sequences are unrelated, components of CE-binding complexes, the yeast core factor (CF) and the human Selectivity Factor 1 (SL1) share functional and structural homology [4][5][6][7]. CF and SL1 are required to recruit the conserved initiation competent Rrn3-Pol I complex to the rDNA promoter [8,9]. Stable recruitment of CF to the rDNA promoter in yeast depends on the 6-subunit upstream activation factor (UAF) binding to the UE [10]. ...
RNA polymerase I (Pol I) synthesizes ribosomal RNA (rRNA) in all eukaryotes, accounting for the major part of transcriptional activity in proliferating cells. Although basal Pol I transcription factors have been characterized in diverse organisms, the molecular basis of the robust rRNA production in vivo remains largely unknown. In S. cerevisiae, the multifunctional Net1 protein was reported to stimulate Pol I transcription. We found that the Pol I-stimulating function can be attributed to the very C-terminal region (CTR) of Net1. The CTR was required for normal cell growth and Pol I recruitment to rRNA genes in vivo and sufficient to promote Pol I transcription in vitro. Similarity with the acidic tail region of mammalian Pol I transcription factor UBF, which could partly functionally substitute for the CTR, suggests conserved roles for CTR-like domains in Pol I transcription from yeast to human.
... According to our model, we expect these factors to bind rDNA tightly and lack any substantial pool of unbound protein in the nucleolus. This view is supported by the low abundance of these proteins (Ghaemmaghami et al., 2003) and by biochemical work which indicated stable association of the CF subunits Rrn6 and Rrn7, as well as UAF, with the rDNA promoter (Keys et al., 1994;Keys, Lee, et al., 1996). In addition, a recent study reported that Fob1, unlike other nucleolar proteins, stayed associated with the rDNA during starvation-induced nucleophagy (Mostofa et al., 2018) supporting the view of persistent rDNA binding by Fob1. ...
The nucleolus is a membraneless organelle of the nucleus and the site of rRNA synthesis, maturation, and assembly into preribosomal particles. The nucleolus, organized around arrays of rRNA genes (rDNA), dissolves during prophase of mitosis in metazoans, when rDNA transcription ceases, and reforms in telophase, when rDNA transcription resumes. No such dissolution and reformation cycle exists in budding yeast, and the precise course of nucleolar segregation remains unclear. By quantitative live-cell imaging, we observed that the yeast nucleolus is reorganized in its protein composition during mitosis. Daughter cells received equal shares of preinitiation factors, which bind the RNA polymerase I promoter and the rDNA binding barrier protein Fob1, but only about one-third of RNA polymerase I and the processing factors Nop56 and Nsr1. The distribution bias was diminished in nonpolar chromosome segregation events observable in dyn1 mutants. Unequal distribution, however, was enhanced by defects in RNA polymerase I, suggesting that rDNA transcription supports nucleolar segregation. Indeed, quantification of pre-rRNA levels indicated ongoing rDNA transcription in yeast mitosis. These data, together with photobleaching experiments to measure nucleolar protein dynamics in anaphase, consolidate a model that explains the differential partitioning of nucleolar components in budding yeast mitosis.
Ribosomal RNAs (rRNAs) are structural components of ribosomes and represent the most abundant cellular RNA fraction. In the yeast Saccharomyces cerevisiae , they account for more than 60 % of the RNA content in a growing cell. The major amount of rRNA is synthesized by RNA polymerase I (Pol I). This enzyme transcribes exclusively the rRNA gene which is tandemly repeated in about 150 copies on chromosome XII. The high number of transcribed rRNA genes, the efficient recruitment of the transcription machinery and the dense packaging of elongating Pol I molecules on the gene ensure that enough rRNA is generated. Specific features of Pol I and of associated factors confer promoter selectivity and both elongation and termination competence. Many excellent reviews exist about the state of research about function and regulation of Pol I and how Pol I initiation complexes are assembled. In this report we focus on the Pol I specific lobe binding subunits which support efficient, error-free, and correctly terminated rRNA synthesis.
Transcription is a highly regulated process that supplies living cells with coding and non-coding RNA molecules. Failure to properly regulate transcription is associated with human pathologies, including cancers. RNA polymerase II is the enzyme complex that synthesizes messenger RNAs that are then translated into proteins. In spite of its complexity, RNA polymerase requires a plethora of general transcription factors to be recruited to the transcription start site as part of a large transcription pre-initiation complex, and to help it gain access to the transcribed strand of the DNA. This chapter reviews the structure and function of these eukaryotic transcription pre-initiation complexes, with a particular emphasis on two of its constituents, the multisubunit complexes TFIID and TFIIH. We also compare the overall architecture of the RNA polymerase II pre-initiation complex with those of RNA polymerases I and III, involved in transcription of ribosomal RNA and non-coding RNAs such as tRNAs and snRNAs, and discuss the general, conserved features that are applicable to all eukaryotic RNA polymerase systems.
Ixr1 is a Saccharomyces cerevisiae HMGB protein that regulates the hypoxic regulon and also controls the expression of other genes involved in the oxidative stress response or re-adaptation of catabolic and anabolic fluxes when oxygen is limiting. Ixr1 also binds with high affinity to cisplatin-DNA adducts and modulates DNA repair. The influence of Ixr1 on transcription in the absence or presence of cisplatin has been analyzed in this work. Ixr1 regulates other transcriptional factors that respond to nutrient availability or extracellular and intracellular stress stimuli, some controlled by the TOR pathway and PKA signaling. Ixr1 controls transcription of ribosomal RNAs and genes encoding ribosomal proteins or involved in ribosome assembly. qPCR, ChIP, and 18S and 25S rRNAs measurement have confirmed this function. Ixr1 binds directly to several promoters of genes related to rRNA transcription and ribosome biogenesis. Cisplatin treatment mimics the effect of IXR1 deletion on rRNA and ribosomal gene transcription, and prevents Ixr1 binding to specific promoters related to these processes.
The gene and protein structure of the mouse UBF (mUBF), a transcription factor for mouse ribosomal RNA gene, have been determined
by cDNA and genomic clones. The unique mUBF gene consists of 21 exons spanning over 13 kb. Two mRNAs coding for mUBF1 and
mUBF2 having 765 a.a. and 728 a.a., respectively, are produced by an alternative splicing of exon 8. It specifies 37 amino
acids constituting a part of the regions homologous to high mobility group proteins (HMG box 2). A human UBF (hUBF) cDNA obtained
by polymerase chain reaction also indicates the presence of two kinds of mRNAs, the shorter form lacking the same regoin as
mUBF2. Comparison of the cDNAs from hUBF and mUBF revealed an unusual conservation of nucleotide sequence in the 3'-terminat
non-coding region. We examined the relative amounts of expression of mUBF1 and mUBF2. The eight tissues studied contained
both molecular species, although mUBF2 was the predominant form of (JBF. The mRNA of mUBF1 was expressed one half of the mUBF2
in quiescent mouse fibroblasts but reached the same amount in growing state.
The synthesis of ribosomes is regulated in response to environmental changes, and this regulation is important in connection with the regulation of growth. This is true for both prokaryotic and eukaryotic organisms. In E. coil, the cellular concentration of ribosomes is roughly proportional to the growth rate except under slow growth conditions; this regulatory feature is called growth-rate-dependent control of ribosome synthesis. Extensive studies of this regulation in E. coli have shown that the regulation acts primarily on the synthesis of rRNA, and that growth-rate-dependent control of ribosomal protein (r-protein) synthesis is achieved indirectly through various autogenous (feedback) repression systems. In eukaryotes, although the synthesis of r-proteins may be regulated directly, and independently of rRNA synthesis, there is no doubt that the synthesis of rRNAs in the nucleolus is of primary importance in overall regulation of ribosome biosynthesis (see reviews, Warner, 1982; Sollner-Webb and Tower, 1986; Sollner-Webb and Mougey, 1991). A few years ago, our laboratory initiated studies on the synthesis of rRNA in the yeast Saccharomyces cerevislae as a model eukaryotic organism, with the eventual goal of understanding molecular events involved in the regulation of ribosome biosynthesis.
This chapter discusses the mechanisms of eukaryotic ribosomal transcription and its regulation. Ribosomal transcription has been studied in an exceptionally wide range of organisms. In eukaryotes, ribosomal gene transcription uses a dedicated set of transcription factors and a specialized ribonucleic acid (RNA) polymerase, the deoxyribonucleic acid (DNA)-dependent RNA polymerase I (RPOI). The resultant precursor-rRNA (pre-rRNA) is neither capped nor polyadenylated and is produced in the nucleolus; a large nuclear structure visible in the light microscope. Moreover, the promotion of ribosomal transcription is generally directed, by a 100- to 150-bp DNA sequence that lies across and, predominantly, upstream of the transcription-initiation site. Promotion requires an activated form of RPOI, the DNA-binding factor upstream transcription factor (UBF), and the TATA binding protein (TBP)I-complex, containing three TATA-box binding protein associated protein (TAF)Is. Upstream binding factor (UBF) binds to both an upstream and an initiation-site promoter element, probably coiling the promoter into a 180-bp loop. This allows the interaction of the TBP,-complex, and subsequent polymerase recruitment. The activity of the promoter is enhanced by a promoter-proximal terminator, by enhancer repeats in the proximal IGS, and by the presence in the IGS of duplicate spacer promoters. Further upstream, other sequences may also modulate transcription.
The DNA sequence of an 8079 bp ClaI fragment located at 40 kb from the centromere on the left arm of chromosome II from Saccharomyces cerevisiae has been determined. Sequence analysis reveals five new open reading frames, tRNA(Gly) and tRNA(Leu) genes as well as sigma and truncated delta elements. The disruption of the three larger open reading frames shows that they are not essential for mitotic growth.
We have previously shown that the TATA-binding protein (TBP) and multiple TBP-associated factors (TAFs) are required for regulated transcriptional initiation by RNA polymerase II. Here we report the biochemical properties of the RNA polymerase I promoter selectivity factor, SL1, and its relationship to TBP. Column chromatography and glycerol gradient sedimentation indicate that a subpopulation of TBP copurifies with SL1 activity. Antibodies directed against TBP efficiently deplete SL1 transcriptional activity, which can be restored with the SL1 fraction but not purified TBP. Thus, TBP is necessary but not sufficient to complement SL1 activity. Analysis of purified SL1 reveals a complex containing TBP and three distinct TAFs. Purified TAFs reconstituted with recombinant TBP complement SL1 activity, and this demonstrates that TBP plus novel associated factors are integral components of SL1. These findings suggest that TBP may be a universal transcription factor and that the TBP-TAF arrangement provides a unifying mechanism for promoter recognition in animal cells.
Using temperature- and proteolytically sensitive derivatives to inactivate the function of the yeast TATA-binding protein (TBP) in vivo, we investigated the requirement of TBP for transcription by the three nuclear RNA polymerases in yeast cells. TBP is required for RNA polymerase II (pol II) transcription from promoters containing conventional TATA elements as well as functionally distinct promoters that lack TATA-like sequences. TBP is also required for transcription of the U6 snRNA and two different tRNA genes mediated by RNA pol III as well as transcription of ribosomal RNA mediated by RNA pol I. For all promoters tested, transcription decreases rapidly and specifically upon inactivation of TBP, strongly suggesting that TBP is directly involved in the transcription process. These observations suggest that TBP is required for transcription of all nuclearly encoded genes in yeast, although distinct molecular mechanisms are probably involved for the three RNA polymerase transcription machineries.