RESUMO
In eukaryotes, each of the more than 100 copies of ribosomal RNA (rRNA) genes exists in either an RNA polymerase I transcribed open chromatin state or a nucleosomal, closed chromatin state. Open rRNA genes guarantee the cell's supply with structural components of the ribosome, whereas closed rRNA genes ensure genomic integrity. We report that the observed balance between open and closed rRNA gene chromatin states in proliferating yeast cells is due to a dynamic equilibrium of transcription-dependent removal and replication-dependent assembly of nucleosomes. Pol I transcription is required for the association of the HMG box protein Hmo1 with open rRNA genes, counteracting replication-independent nucleosome deposition and maintaining the open rRNA gene chromatin state outside of S phase. The findings indicate that the opposing effects of replication and transcription lead to a de novo establishment of chromatin states for rRNA genes during each cell cycle.
Assuntos
Cromatina/metabolismo , Genes de RNAr , Saccharomyces cerevisiae/citologia , Ciclo Celular , Replicação do DNA , DNA Ribossômico/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , RNA Polimerase I/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
Elongating nuclear RNA polymerases (Pols) frequently pause, backtrack, and are then reactivated by endonucleolytic cleavage. Pol backtracking and RNA cleavage are also crucial for proofreading, which contributes to transcription fidelity. RNA polymerase I (Pol I) of the yeast Saccharomyces cerevisiae synthesizes exclusively 35S rRNA, the precursor transcript of mature ribosomal 5.8S, 18S, and 25S rRNA. Pol I contains the specific heterodimeric subunits Rpa34.5/49 and subunit Rpa12.2, which have been implicated in RNA cleavage and elongation activity, respectively. These subunits are associated with the Pol I lobe structure and encompass different structural domains, but the contribution of these domains to RNA elongation is unclear. Here, we used Pol I mutants or reconstituted Pol I enzymes to study the effects of these subunits and/or their distinct domains on RNA cleavage, backtracking, and transcription fidelity in defined in vitro systems. Our findings suggest that the presence of the intact C-terminal domain of Rpa12.2 is sufficient to support the cleavage reaction, but that the N-terminal domains of Rpa12.2 and the heterodimer facilitate backtracking and RNA cleavage. Since both N-terminal and C-terminal domains of Rpa12.2 were also required to faithfully incorporate NTPs in the growing RNA chain, efficient backtracking and RNA cleavage might be a prerequisite for transcription fidelity. We propose that RNA Pols containing efficient RNA cleavage activity are able to add and remove nucleotides until the matching nucleotide supports RNA chain elongation, whereas cleavage-deficient enzymes can escape this proofreading process by incorporating incorrect nucleotides.
Assuntos
RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Nucleotídeos , RNA , Clivagem do RNA , Saccharomyces cerevisiae/metabolismo , Transcrição GênicaRESUMO
The ribosomal RNA precursor (pre-rRNA) comprises three of the four ribosomal RNAs and is synthesized by RNA polymerase (Pol) I. Here, we describe the mechanisms of Pol I transcription in human cells with a focus on recent insights gained from structure-function analyses. The comparison of Pol I-specific structural and functional features with those of other Pols and with the excessively studied yeast system distinguishes organism-specific from general traits. We explain the organization of the genomic rDNA loci in human cells, describe the Pol I transcription cycle regarding structural changes in the enzyme and the roles of human Pol I subunits, and depict human rDNA transcription factors and their function on a mechanistic level. We disentangle information gained by direct investigation from what had apparently been deduced from studies of the yeast enzymes. Finally, we provide information about how Pol I mutations may contribute to developmental diseases, and why Pol I is a target for new cancer treatment strategies, since increased rRNA synthesis was correlated with rapidly expanding cell populations.
Assuntos
Precursores de RNA , RNA Ribossômico , Humanos , RNA Ribossômico/genética , Precursores de RNA/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , RNA Polimerase I/genética , RNA Polimerase I/química , RNA Polimerase I/metabolismo , DNA Ribossômico/genéticaRESUMO
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 60â¯% 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.
Assuntos
RNA Polimerase I , Saccharomyces cerevisiae , RNA Polimerase I/química , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Ribossomos/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Sequência de Aminoácidos , Proteínas de Ciclo Celular/genética , Sequência Conservada , Humanos , Proteínas Nucleares/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/química , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Ribossômico/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Deleção de Sequência , Transcrição GênicaRESUMO
Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.
Assuntos
Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , RNA Polimerase I/genética , DNA Ribossômico/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Precursores de RNA/genética , RNA Ribossômico , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
RNA polymerase I (Pol I) is a highly efficient enzyme specialized in synthesizing most ribosomal RNAs. After nucleosome deposition at each round of rDNA replication, the Pol I transcription machinery has to deal with nucleosomal barriers. It has been suggested that Pol I-associated factors facilitate chromatin transcription, but it is unknown whether Pol I has an intrinsic capacity to transcribe through nucleosomes. Here, we used in vitro transcription assays to study purified WT and mutant Pol I variants from the yeast Saccharomyces cerevisiae and compare their abilities to pass a nucleosomal barrier with those of yeast Pol II and Pol III. Under identical conditions, purified Pol I and Pol III, but not Pol II, could transcribe nucleosomal templates. Pol I mutants lacking either the heterodimeric subunit Rpa34.5/Rpa49 or the C-terminal part of the specific subunit Rpa12.2 showed a lower processivity on naked DNA templates, which was even more reduced in the presence of a nucleosome. Our findings suggest that the lobe-binding subunits Rpa34.5/Rpa49 and Rpa12.2 facilitate passage through nucleosomes, suggesting possible cooperation among these subunits. We discuss the contribution of Pol I-specific subunit domains to efficient Pol I passage through nucleosomes in the context of transcription rate and processivity.
Assuntos
Cromatina/metabolismo , Nucleossomos/metabolismo , RNA Polimerase III/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase I/metabolismo , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Cromatina/genética , Replicação do DNA , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Nucleossomos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Polimerase I/química , RNA Polimerase I/genética , RNA Polimerase II/química , RNA Polimerase II/genética , RNA Polimerase III/química , RNA Polimerase III/genética , Ribossomos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.
Assuntos
Quelantes/farmacologia , Inibidores Enzimáticos/farmacologia , Metaloproteases/antagonistas & inibidores , Transativadores/antagonistas & inibidores , Zinco/química , Quelantes/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Células HeLa , Humanos , Metaloproteases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacologia , Relação Estrutura-Atividade , Transativadores/metabolismoRESUMO
In all living organisms, the flow of genetic information is a two-step process: first DNA is transcribed into RNA, which is subsequently used as template for protein synthesis during translation. In bacteria, archaea and eukaryotes, transcription is carried out by multi-subunit RNA polymerases (RNAPs) sharing a conserved architecture of the RNAP core. RNAPs catalyse the highly accurate polymerisation of RNA from NTP building blocks, utilising DNA as template, being assisted by transcription factors during the initiation, elongation and termination phase of transcription. The complexity of this highly dynamic process is reflected in the intricate network of protein-protein and protein-nucleic acid interactions in transcription complexes and the substantial conformational changes of the RNAP as it progresses through the transcription cycle.In this chapter, we will first briefly describe the early work that led to the discovery of multisubunit RNAPs. We will then discuss the three-dimensional organisation of RNAPs from the bacterial, archaeal and eukaryotic domains of life, highlighting the conserved nature, but also the domain-specific features of the transcriptional apparatus. Another section will focus on transcription factors and their role in regulating the RNA polymerase throughout the different phases of the transcription cycle. This includes a discussion of the molecular mechanisms and dynamic events that govern transcription initiation, elongation and termination.
Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Gênica , Animais , Archaea/enzimologia , Bactérias/enzimologia , Eucariotos/enzimologia , Humanos , Fatores de Transcrição/metabolismoRESUMO
Several DNA cis-elements and trans-acting factors were described to be involved in transcription termination and to release the elongating RNA polymerases from their templates. Different models for the molecular mechanism of transcription termination have been suggested for eukaryotic RNA polymerase I (Pol I) from results of in vitro and in vivo experiments. To analyse the molecular requirements for yeast RNA Pol I termination, an in vivo approach was used in which efficient termination resulted in growth inhibition. This led to the identification of a Myb-like protein, Ydr026c, as bona fide termination factor, now designated Nsi1 (NTS1 silencing protein 1), since it was very recently described as silencing factor of ribosomal DNA. Possible Nsi1 functions in regard to the mechanism of transcription termination are discussed.
Assuntos
Proteínas de Ligação a DNA/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transcrição Gênica , Sequência de Aminoácidos , Dados de Sequência Molecular , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Homologia de Sequência de AminoácidosRESUMO
Chromatin is the template for replication and transcription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purification of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae. Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry. We present and discuss the proteomic data sets obtained for chromatin in different functional states. The native chromatin was further amenable to electron microscopy analysis yielding information about nucleosome occupancy and positioning at the single-molecule level. We also provide evidence that chromatin from virtually every single copy genomic locus of interest can be purified and analyzed by this technique.
Assuntos
Cromossomos Fúngicos/química , Saccharomyces cerevisiae/genética , Fosfatase Ácida/genética , DNA Ribossômico/química , DNA Ribossômico/isolamento & purificação , Genômica/métodos , Histonas/metabolismo , Espectrometria de Massas , Nucleossomos/química , Regiões Promotoras Genéticas , Proteoma/isolamento & purificação , RNA Ribossômico 5S/química , RNA Ribossômico 5S/ultraestrutura , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificaçãoRESUMO
Chromosome condensation and the global repression of gene transcription are features of mitosis in most eukaryotes. The logic behind this phenomenon is that chromosome condensation prevents the activity of RNA polymerases. In budding yeast, however, transcription was proposed to be continuous during mitosis. Here we show that Cdc14, a protein phosphatase required for nucleolar segregation and mitotic exit, inhibits transcription of yeast ribosomal genes (rDNA) during anaphase. The phosphatase activity of Cdc14 is required for RNA polymerase I (Pol I) inhibition in vitro and in vivo. Moreover Cdc14-dependent inhibition involves nucleolar exclusion of Pol I subunits. We demonstrate that transcription inhibition is necessary for complete chromosome disjunction, because ribosomal RNA (rRNA) transcripts block condensin binding to rDNA, and show that bypassing the role of Cdc14 in nucleolar segregation requires in vivo degradation of nascent transcripts. Our results show that transcription interferes with chromosome condensation, not the reverse. We conclude that budding yeast, like most eukaryotes, inhibit Pol I transcription before segregation as a prerequisite for chromosome condensation and faithful genome separation.
Assuntos
Anáfase/fisiologia , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , RNA Polimerase I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transcrição Gênica/fisiologia , Adenosina Trifosfatases/metabolismo , Segregação de Cromossomos , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Ligação Proteica/fisiologia , RNA Fúngico/metabolismo , RNA Ribossômico/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic ribosome biogenesis requires more than 150 auxiliary proteins, which transiently interact with pre-ribosomal particles. Previous studies suggest that several of these biogenesis factors function together as modules. Using a heterologous expression system, we show that the large ribosomal subunit (LSU) biogenesis factor Noc1p of Saccharomyces cerevisiae can simultaneously interact with the LSU biogenesis factor Noc2p and Rrp5p, a factor required for biogenesis of the large and the small ribosomal subunit. Proteome analysis of RNA polymerase-I-associated chromatin and chromatin immunopurification experiments indicated that all members of this protein module and a specific set of LSU biogenesis factors are co-transcriptionally recruited to nascent ribosomal RNA (rRNA) precursors in yeast cells. Further ex vivo analyses showed that all module members predominantly interact with early pre-LSU particles after the initial pre-rRNA processing events have occurred. In yeast strains depleted of Noc1p, Noc2p or Rrp5p, levels of the major LSU pre-rRNAs decreased and the respective other module members were associated with accumulating aberrant rRNA fragments. Therefore, we conclude that the module exhibits several binding interfaces with pre-ribosomes. Taken together, our results suggest a co- and post-transcriptional role of the yeast Rrp5p-Noc1p-Noc2p module in the structural organization of early LSU precursors protecting them from non-productive RNase activity.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Proteínas Nucleares/química , Proteínas de Ligação a RNA/química , Proteínas Recombinantes/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Transcrição GênicaRESUMO
Eukaryotic transcription of ribosomal RNAs (rRNAs) by RNA polymerase I can account for more than half of the total cellular transcripts depending on organism and growth condition. To support this level of expression, eukaryotic rRNA genes are present in multiple copies. Interestingly, these genes co-exist in different chromatin states that may differ significantly in their nucleosome content and generally correlate well with transcriptional activity. Here we review how these chromatin states have been discovered and characterized focusing particularly on their structural protein components. The establishment and maintenance of rRNA gene chromatin states and their impact on rRNA synthesis are discussed. This article is part of a Special Issue entitled: Transcription by Odd Pols.
Assuntos
Cromatina/química , DNA Ribossômico/química , Transcrição Gênica , Animais , Cromatina/metabolismo , DNA Ribossômico/metabolismo , Epigênese Genética , Loci Gênicos , Humanos , RNA Polimerase I/metabolismo , RNA Ribossômico/biossíntese , RNA Ribossômico/genéticaRESUMO
The synthesis of ribosomal RNA (rRNA) precursor molecules by RNA polymerase I (Pol I) terminates with the dissociation of the protein-DNA-RNA ternary complex. Based on in vitro results the mechanism of Pol I termination appeared initially to be rather conserved and simple until this process was more thoroughly re-investigated in vivo. A picture emerged that Pol I termination seems to be connected to co-transcriptional processing, re-initiation of transcription and, possibly, other processes downstream of Pol I transcription units. In this article, our current understanding of the mechanism of Pol I termination and how this process might be implicated in other biological processes in yeast and mammals is summarized and discussed. This article is part of a Special Issue entitled: Transcription by Odd Pols.
Assuntos
RNA Polimerase I/metabolismo , Terminação da Transcrição Genética , Animais , Humanos , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Precursores de RNA/biossíntese , Precursores de RNA/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Fatores de Complexo Ternário/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
RpS0/uS2, rpS2/uS5, and rpS21/eS21 form a cluster of ribosomal proteins (S0-cluster) at the head-body junction near the central pseudoknot of eukaryotic small ribosomal subunits (SSU). Previous work in yeast indicated that S0-cluster assembly is required for the stabilisation and maturation of SSU precursors at specific post-nucleolar stages. Here, we analysed the role of S0-cluster formation for rRNA folding. Structures of SSU precursors isolated from yeast S0-cluster expression mutants or control strains were analysed by cryogenic electron microscopy. The obtained resolution was sufficient to detect individual 2'-O-methyl RNA modifications using an unbiased scoring approach. The data show how S0-cluster formation enables the initial recruitment of the pre-rRNA processing factor Nob1 in yeast. Furthermore, they reveal hierarchical effects on the pre-rRNA folding pathway, including the final maturation of the central pseudoknot. Based on these structural insights we discuss how formation of the S0-cluster determines at this early cytoplasmic assembly checkpoint if SSU precursors further mature or are degraded.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores/química , Subunidades Ribossômicas Menores/metabolismo , Precursores de RNA/genética , Precursores de RNA/química , RNA Ribossômico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Conformação de Ácido NucleicoRESUMO
Formation of eukaryotic ribosomes requires more than 150 biogenesis factors which transiently interact with the nascent ribosomal subunits. Previously, many pre-ribosomal intermediates could be distinguished by their protein composition and rRNA precursor (pre-rRNA) content. We purified complexes of ribosome biogenesis factors from yeast cells in which de novo synthesis of rRNA precursors was down-regulated by genetic means. We compared the protein composition of these largely pre-rRNA free assemblies with the one of analogous pre-ribosomal preparations by semi-quantitative mass spectrometry. The experimental setup minimizes the possibility that the analysed pre-rRNA free protein modules were derived from (partially) disrupted pre-ribosomal particles and provides thereby strong evidence for their pre-ribosome independent existence. In support of the validity of this approach (i) the predicted composition of the analysed protein modules was in agreement with previously described rRNA-free complexes and (ii) in most of the cases we could identify new candidate members of reported protein modules. An unexpected outcome of these analyses was that free large ribosomal subunits are associated with a specific set of ribosome biogenesis factors in cells where neo-production of nascent ribosomes was blocked. The data presented strengthen the idea that assembly of eukaryotic pre-ribosomal particles can result from transient association of distinct building blocks.
Assuntos
Proteínas Fúngicas/análise , Proteínas Ribossômicas/análise , Ribossomos/química , Proteínas Fúngicas/metabolismo , Precursores de RNA/metabolismo , RNA Fúngico/biossíntese , RNA Fúngico/metabolismo , RNA Ribossômico/biossíntese , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Leveduras/genéticaRESUMO
Ribosome biogenesis is tightly linked to cellular growth. A crucial step in the regulation of ribosomal RNA (rRNA) gene transcription is the formation of the complex between RNA polymerase I (Pol I) and the Pol I-dependent transcription factor Rrn3p. We found that TOR inactivation leads to proteasome-dependent degradation of Rrn3p and a strong reduction in initiation competent Pol I-Rrn3p complexes affecting yeast rRNA gene transcription. Using a mutant expressing non-degradable Rrn3p or a strain in which defined endogenous Rrn3p levels can be adjusted by the Tet-off system, we can demonstrate that Rrn3p levels influence the number of Pol I-Rrn3p complexes and consequently rRNA gene transcription. However, our analysis reveals that the dramatic reduction of rRNA synthesis in the immediate cellular response to impaired TOR signalling cannot be explained by the simple down-regulation of Rrn3p and Pol I-Rrn3p levels.