scholarly journals RNA Polymerase I-Promoted HIS4Expression Yields Uncapped, Polyadenylated mRNA That Is Unstable and Inefficiently Translated in Saccharomyces cerevisiae

1998 ◽  
Vol 18 (2) ◽  
pp. 665-675 ◽  
Author(s):  
Hsiu-Jung Lo ◽  
Han-Kuei Huang ◽  
Thomas F. Donahue
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Mutant Form ◽  
Wild Type ◽  
Pol I ◽  
Chromosome Iii ◽  
Polymerase I

ABSTRACT The HIS4 gene in Saccharomyces cerevisiaewas put under the transcriptional control of RNA polymerase I to determine the in vivo consequences on mRNA processing and gene expression. This gene, referred to as rhis4, was substituted for the normal HIS4 gene on chromosome III. Therhis4 gene transcribes two mRNAs, of which each initiates at the polymerase (pol) I transcription initiation site. One transcript, rhis4s, is similar in size to the wild-typeHIS4 mRNA. Its 3′ end maps to the HIS4 3′ noncoding region, and it is polyadenylated. The second transcript,rhis4l, is bicistronic. It encodes the HIS4coding region and a second open reading frame, YCL184, that is located downstream of the HIS4 gene and is predicted to be transcribed in the same direction as HIS4 on chromosome III. The 3′ end of rhis4l maps to the predicted 3′ end of the YCL184 gene and is also polyadenylated. Based on in vivo labeling experiments, the rhis4 gene appears to be more actively transcribed than the wild-type HIS4 gene despite the near equivalence of the steady-state levels of mRNAs produced from each gene. This finding indicated that rhis4mRNAs are rapidly degraded, presumably due to the lack of a cap structure at the 5′ end of the mRNA. Consistent with this interpretation, a mutant form of XRN1, which encodes a 5′-3′ exonuclease, was identified as an extragenic suppressor that increases the half-life of rhis4 mRNA, leading to a 10-fold increase in steady-state mRNA levels compared to the wild-typeHIS4 mRNA level. This increase is dependent on pol I transcription. Immunoprecipitation by anticap antiserum suggests that the majority of rhis4 mRNA produced is capless. In addition, we quantitated the level of His4 protein in a rhis4 xrn1Δ genetic background. This analysis indicates that capless mRNA is translated at less than 10% of the level of translation of capped HIS4 mRNA. Our data indicate that polyadenylation of mRNA in yeast occurs despite HIS4 being transcribed by RNA polymerase I, and the 5′ cap confers stability to mRNA and affords the ability of mRNA to be translated efficiently in vivo.

scholarly journals Downstream sequence-dependent RNA cleavage and pausing by RNA polymerase I

2019 ◽  
Vol 295 (5) ◽  
pp. 1288-1299 ◽  
Author(s):  
Catherine E. Scull ◽  
Andrew M. Clarke ◽  
Aaron L. Lucius ◽  
David Alan Schneider
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Dna Sequence ◽  
Transcription Elongation ◽  
Gc Content ◽  
Rna Polymerase I ◽  
Pol I ◽  
Polymerase I

The sequence of the DNA template has long been thought to influence the rate of transcription by DNA-dependent RNA polymerases, but the influence of DNA sequence on transcription elongation properties of eukaryotic RNA polymerase I (Pol I) from Saccharomyces cerevisiae has not been defined. In this study, we observe changes in dinucleotide production, transcription elongation complex stability, and Pol I pausing in vitro in response to downstream DNA. In vitro studies demonstrate that AT-rich downstream DNA enhances pausing by Pol I and inhibits Pol I nucleolytic cleavage activity. Analysis of Pol I native elongating transcript sequencing data in Saccharomyces cerevisiae suggests that these downstream sequence elements influence Pol I in vivo. Native elongating transcript sequencing studies reveal that Pol I occupancy increases as downstream AT content increases and decreases as downstream GC content increases. Collectively, these data demonstrate that the downstream DNA sequence directly impacts the kinetics of transcription elongation prior to the sequence entering the active site of Pol I both in vivo and in vitro.

scholarly journals The relationship between the activity of DNA-dependent RNA polymerase I and the rate of synthesis of rRNA in hepatoma cells in culture

1981 ◽  
Vol 194 (1) ◽  
pp. 43-51 ◽  
Author(s):  
E A Thompson ◽  
R H Keith ◽  
A H Cavanaugh ◽  
K M Wood
Keyword(s):  
Steady State ◽  
Rna Polymerase ◽  
Cell Lines ◽  
Rna Polymerase I ◽  
28S Rrna ◽  
Pol I ◽  
Cell Enzyme ◽  
Ribonucleoside Triphosphate ◽  
Polymerase I ◽  
The Relationship

Cell culture lines were established from the transplantable mouse hepatomas H6 and H129. Both cell lines had a doubling time about 30 h when maintained in medium containing 5% foetal bovine serum. H6 cells contained about 3-4 times more DNA-dependent RNA polymerase I (Pol I; ribonucleoside triphosphate–RNA nucleotidyltransferase, EC 2.7.7.6) than did H129 cells. Moreover, the H6-cell enzyme was more heat-labile than that from H129 cells. Steady-state contents of 28S rRNA were measured in both cell lines. Exponentially growing cultures of H6 cells contained about 6.5pg of 28S rRNA/cell, and similar cultures of H129 cells contained about 5.8pg/cell. Stationary cultures of both cell lines contained about 2pg of 28S rRNA/cell. By two different techniques, the half-time for turnover of 28S rRNA was estimated to be 16-17h for both H6 and H129 cells. Knowing the turnover rate and the steady-state concentration, one may calculate that both H6 and H129 cells synthesize 28S rRNA at a rate of about 0.25 pg/h per cell. The amount of template-bound Pol I activity was similar in nuclei isolated from H6 and H129 cell cultures. These data indicate that, although H6 cells contained 3-4 times more Pol I than did H129 cells, both cell lines synthesized rRNA at about the same rate.

Cloning and characterization of SRP1, a suppressor of temperature-sensitive RNA polymerase I mutations, in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (12) ◽  
pp. 5640-5651
Author(s):  
R Yano ◽  
M Oakes ◽  
M Yamaghishi ◽  
J A Dodd ◽  
M Nomura
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Rna Polymerase ◽  
Binding Domain ◽  
Rna Polymerase I ◽  
Zinc Binding ◽  
Temperature Sensitive ◽  
Pol I ◽  
Polymerase I ◽  
Zinc Binding Domain

The SRP1-1 mutation is an allele-specific dominant suppressor of temperature-sensitive mutations in the zinc-binding domain of the A190 subunit of Saccharomyces cerevisiae RNA polymerase I (Pol I). We found that it also suppresses temperature-sensitive mutations in the zinc-binding domain of the Pol I A135 subunit. This domain had been suggested to be in physical proximity to the A190 zinc-binding domain. We have cloned the SRP1 gene and determined its nucleotide sequence. The gene encodes a protein of 542 amino acids consisting of three domains: the central domain, which is composed of eight (degenerate) 42-amino-acid contiguous tandem repeats, and the surrounding N-terminal and C-terminal domains, both of which contain clusters of acidic and basic amino acids and are very hydrophilic. The mutational alteration (P219Q) responsible for the suppression was found to be in the central domain. Using antibody against the SRP1 protein, we have found that SRP1 is mainly localized at the periphery of the nucleus, apparently more concentrated in certain regions, as suggested by a punctate pattern in immunofluorescence microscopy. We suggest that SRP1 is a component of a larger macromolecular complex associated with the nuclear envelope and interacts with Pol I either directly or indirectly through other components in the structure containing SRP1.

scholarly journals The dynamic assembly of distinct RNA polymerase I complexes modulates rDNA transcription

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Eva Torreira ◽  
Jaime Alegrio Louro ◽  
Irene Pazos ◽  
Noelia González-Polo ◽  
David Gil-Carton ◽  
...  
Keyword(s):  
Cell Growth ◽  
Rna Polymerase ◽  
Ribosome Biogenesis ◽  
Mutational Analysis ◽  
Rna Polymerase I ◽  
Initiation Factor ◽  
Rdna Transcription ◽  
Pol I ◽  
Polymerase I

Cell growth requires synthesis of ribosomal RNA by RNA polymerase I (Pol I). Binding of initiation factor Rrn3 activates Pol I, fostering recruitment to ribosomal DNA promoters. This fundamental process must be precisely regulated to satisfy cell needs at any time. We present in vivo evidence that, when growth is arrested by nutrient deprivation, cells induce rapid clearance of Pol I–Rrn3 complexes, followed by the assembly of inactive Pol I homodimers. This dual repressive mechanism reverts upon nutrient addition, thus restoring cell growth. Moreover, Pol I dimers also form after inhibition of either ribosome biogenesis or protein synthesis. Our mutational analysis, based on the electron cryomicroscopy structures of monomeric Pol I alone and in complex with Rrn3, underscores the central role of subunits A43 and A14 in the regulation of differential Pol I complexes assembly and subsequent promoter association.

scholarly journals Casein Kinase 2 Associates with Initiation-Competent RNA Polymerase I and Has Multiple Roles in Ribosomal DNA Transcription

2006 ◽  
Vol 26 (16) ◽  
pp. 5957-5968 ◽  
Author(s):  
Tatiana B. Panova ◽  
Kostya I. Panov ◽  
Jackie Russell ◽  
Joost C. B. M. Zomerdijk
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Casein Kinase ◽  
Casein Kinase 2 ◽  
Rna Polymerase I ◽  
Positive Effects ◽  
Pol I ◽  
Polymerase I

ABSTRACT Mammalian RNA polymerase I (Pol I) complexes contain a number of associated factors, some with undefined regulatory roles in transcription. We demonstrate that casein kinase 2 (CK2) in human cells is associated specifically only with the initiation-competent Pol Iβ isoform and not with Pol Iα. Chromatin immunoprecipitation analysis places CK2 at the ribosomal DNA (rDNA) promoter in vivo. Pol Iβ-associated CK2 can phosphorylate topoisomerase IIα in Pol Iβ, activator upstream binding factor (UBF), and selectivity factor 1 (SL1) subunit TAFI110. A potent and selective CK2 inhibitor, 3,8-dibromo-7-hydroxy-4-methylchromen-2-one, limits in vitro transcription to a single round, suggesting a role for CK2 in reinitiation. Phosphorylation of UBF by CK2 increases SL1-dependent stabilization of UBF at the rDNA promoter, providing a molecular mechanism for the stimulatory effect of CK2 on UBF activation of transcription. These positive effects of CK2 in Pol I transcription contrast to that wrought by CK2 phosphorylation of TAFI110, which prevents SL1 binding to rDNA, thereby abrogating the ability of SL1 to nucleate preinitiation complex (PIC) formation. Thus, CK2 has the potential to regulate Pol I transcription at multiple levels, in PIC formation, activation, and reinitiation of transcription.

scholarly journals RNA Polymerase I Transcription Silences Noncoding RNAs at the Ribosomal DNA Locus in Saccharomyces cerevisiae

2009 ◽  
Vol 9 (2) ◽  
pp. 325-335 ◽  
Author(s):  
Elisa Cesarini ◽  
Francesca Romana Mariotti ◽  
Francesco Cioci ◽  
Giorgio Camilloni
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Ribosomal Dna ◽  
Rna Polymerase Iii ◽  
Noncoding Rnas ◽  
Rdna Locus ◽  
Rna Polymerase I ◽  
Pol Ii ◽  
Pol I ◽  
Polymerase I

ABSTRACT In Saccharomyces cerevisiae the repeated units of the ribosomal locus, transcribed by RNA polymerase I (Pol I), are interrupted by nontranscribed spacers (NTSs). These NTS regions are transcribed by RNA polymerase III to synthesize 5S RNA and by RNA polymerase II (Pol II) to synthesize, at low levels, noncoding RNAs (ncRNAs). While transcription of both RNA polymerase I and III is highly characterized, at the ribosomal DNA (rDNA) locus only a few studies have been performed on Pol II, whose repression correlates with the SIR2-dependent silencing. The involvement of both chromatin organization and Pol I transcription has been proposed, and peculiar chromatin structures might justify “ribosomal” Pol II silencing. Reporter genes inserted within the rDNA units have been employed for these studies. We studied, in the natural context, yeast mutants differing in Pol I transcription in order to find whether correlations exist between Pol I transcription and Pol II ncRNA production. Here, we demonstrate that silencing at the rDNA locus represses ncRNAs with a strength inversely proportional to Pol I transcription. Moreover, localized regions of histone hyperacetylation appear in cryptic promoter elements when Pol II is active and in the coding region when Pol I is functional; in addition, DNA topoisomerase I site-specific activity follows RNA polymerase I transcription. The repression of ncRNAs at the rDNA locus, in response to RNA polymerase I transcription, could represent a physiological circuit control whose mechanism involves modification of histone acetylation.

scholarly journals Genetic analyses led to the discovery of a super-active mutant of the RNA polymerase I

2018 ◽  
Author(s):  
Tommy Darrière ◽  
Michael Pilsl ◽  
Marie-Kerguelen Sarthou ◽  
Adrien Chauvier ◽  
Titouan Genty ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Growth Defect ◽  
Pol I ◽  
Polymerase I ◽  
Extragenic Suppressors

AbstractMost 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 wild-type 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.Author summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

scholarly journals Regulation of Ribosomal RNA Production by RNA Polymerase I: Does Elongation Come First?

2012 ◽  
Vol 2012 ◽  
pp. 1-13 ◽  
Author(s):  
Benjamin Albert ◽  
Jorge Perez-Fernandez ◽  
Isabelle Léger-Silvestre ◽  
Olivier Gadal
Keyword(s):  
Rna Polymerase ◽  
Ribosomal Rna ◽  
Chromatin Structure ◽  
Rna Polymerase I ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Elongation Factors ◽  
Pol I ◽  
Polymerase I

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure in vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo.

scholarly journals Cross Talk between tRNA and rRNA Synthesis inSaccharomyces cerevisiae

2001 ◽  
Vol 21 (1) ◽  
pp. 189-195 ◽  
Author(s):  
Jean-François Briand ◽  
Francisco Navarro ◽  
Olivier Gadal ◽  
Pierre Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Northern Blot Hybridization ◽  
Permissive Temperature ◽  
Wild Type ◽  
Polymerase Iii ◽  
Polymerase I

ABSTRACT Temperature-sensitive RNA polymerase III (rpc160-112and rpc160-270) mutants were analyzed for the synthesis of tRNAs and rRNAs in vivo, using a double-isotopic-labeling technique in which cells are pulse-labeled with [33P]orthophosphate and coextracted with [3H]uracil-labeled wild-type cells. Individual RNA species were monitored by Northern blot hybridization or amplified by reverse transcription. These mutants impaired the synthesis of RNA polymerase III transcripts with little or no influence on mRNA synthesis but also largely turned off the formation of the 25S, 18S, and 5.8S mature rRNA species derived from the common 35S transcript produced by RNA polymerase I. In the rpc160-270mutant, this parallel inhibition of tRNA and rRNA synthesis also occurred at the permissive temperature (25°C) and correlated with an accumulation of 20S pre-rRNA. In the rpc160-112 mutant, inhibition of rRNA synthesis and the accumulation of 20S pre-rRNA were found only at 37°C. The steady-state rRNA/tRNA ratio of these mutants reflected their tRNA and rRNA synthesis pattern: therpc160-112 mutant had the threefold shortage in tRNA expected from its preferential defect in tRNA synthesis at 25°C, whereas rpc160-270 cells completely adjusted their rRNA/tRNA ratio down to a wild-type level, consistent with the tight coupling of tRNA and rRNA synthesis in vivo. Finally, an RNA polymerase I (rpa190-2) mutant grown at the permissive temperature had an enhanced level of pre-tRNA, suggesting the existence of a physiological coupling between rRNA synthesis and pre-tRNA processing.

scholarly journals Saccharomyces cerevisiae RNA Polymerase I Terminates Transcription at the Reb1 Terminator In Vivo

1999 ◽  
Vol 19 (11) ◽  
pp. 7369-7376 ◽  
Author(s):  
Ronald H. Reeder ◽  
Palmira Guevara ◽  
Judith G. Roan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase I ◽  
Rnase Iii ◽  
S1 Nuclease ◽  
Nuclease Mapping ◽  
Fail Safe ◽  
Polymerase I

ABSTRACT We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3′ end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250. Inactivation of Rnt1p (an RNase III involved in processing the 3′ end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies.

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