Characterization of the Components of Reconstituted Saccharomyces cerevisiae RNA Polymerase I Transcription Complexes

Riggs, Daniel L.; Peterson, Cheryl L.; Wickham, J. Quyen; Miller, Letrisa M.; Clarke, Eileen M. W.; Crowell, John A.; Sergere, Jean-Christophe

doi:10.1074/jbc.270.11.6205

Cited by 21 publications

(25 citation statements)

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“…These cultures had generation times of 1.5, 3, 5, and 8 h, respectively. The cells were harvested, and RNAP I and III transcription extracts (low salt pellets and supernatants) were prepared as described previously (36). The levels of specific RNAP I and III transcription were analyzed in vitro using a 35 S rDNA (to assay RNAP I), 5 S rDNA, or tDNA template.…”

Section: Resultsmentioning

confidence: 99%

“…Chromatography of the Transcription Extracts and Characterization of the Fractions-The protocols for cell breakage, extract preparation, Q chromatography, and transcription assays have been previously described (36). For the chromatography of the RNAP III factors the "low salt supernatant" was chromatographed on a Q column developed with a 50 -700 mM KCl gradient.…”

Section: Methodsmentioning

confidence: 99%

“…Despite these advantages, virtually all of the work in yeast has been restricted to in vivo analysis, largely due to the technical difficulties of isolating RNAP I and III transcription extracts from small quantities of cells. To facili-tate the in vitro analysis of stable RNA transcription, we recently developed a method for the preparation of both RNAP I and RNAP III (5 S rDNA and tDNA) transcription extracts from less than 1 g of cells (36). This protocol minimizes the chance of inactivation due to trivial reasons, since no column chromatography is involved, and only at the last step is the RNAP I extract separated from the RNAP III extract.…”

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confidence: 99%

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Regulation of the RNA Polymerase I and III Transcription Systems in Response to Growth Conditions

Clarke

Peterson

Brainard

et al. 1996

Journal of Biological Chemistry

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To better understand the mechanisms that regulate stable RNA synthesis, we have analyzed the RNA polymerase I and III transcriptional activities of extracts isolated from cells propagated under a variety of conditions. Under balanced growth conditions the levels of both RNA polymerase I-and III-specific transcription increased proportionally with growth rate. Upon nutritional starvation, RNA polymerase I transcription rapidly declined, followed by 5 S rDNA and eventually tDNA transcription. Transcriptional activities in extracts were restored when the nongrowing cultures were resuspended in fresh medium, although growth did not resume. The differential expression of 5 S rDNA and tDNA genes in extracts prepared from cells subjected to partial starvation was traced to a 5 S rDNA-specific inhibitor and not to a defect in any RNA polymerase III transcription factor. Characterization of this inhibitor indicated that it was not 5 S rRNA. It was sensitive to phenol extraction and resistant to RNase, and its target did not appear to be transcription factor IIIA. Not all treatments that slowed or stopped growth down-regulated the stable RNA transcription apparatus. Cells that have been subjected to either energy starvation or cycloheximide treatment still retain the ability to synthesize stable RNA in vitro, suggesting the presence of alternative regulatory mechanisms.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Regulation of the RNA Polymerase I and III Transcription Systems in Response to Growth Conditions

Clarke

Peterson

Brainard

et al. 1996

Journal of Biological Chemistry

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“…Growth-rate regulation of rRNA gene expression has been observed in organisms as evolutionarily diverse as yeast (9,23), Acanthamoeba (24), and mammals (15,17,(25)(26)(27). The identification of a human homolog of a yeast transcription factor that …”

Section: Discussionmentioning

confidence: 99%

RNA polymerase I transcription factor Rrn3 is functionally conserved between yeast and human

Moorefield

Greene

Reeder

2000

Proc. Natl. Acad. Sci. U.S.A.

105

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We have cloned a human cDNA that is related to the RNA polymerase I transcription factor Rrn3 of Saccharomyces cerevisiae. The recombinant human protein displays both sequence similarity and immunological crossreactivity to yeast Rrn3 and is capable of rescuing a yeast strain carrying a disruption of the RRN3 gene in vivo. Point mutation of an amino acid that is conserved between the yeast and human proteins compromises the function of each factor, confirming that the observed sequence similarity is functionally significant. Rrn3 is the first RNA polymerase I-specific transcription factor shown to be functionally conserved between yeast and mammals, suggesting that at least one mechanism that regulates ribosomal RNA synthesis is conserved among eukaryotes.RRN3 ͉ ribosomal RNA ͉ growth control ͉ Saccharomyces cerevisiae E ukaryotic ribosomal RNA (rRNA) genes are transcribed by an enzyme solely dedicated to that purpose, RNA polymerase I (pol I), and their expression is coordinated with cellular growth rate. When cell growth is impaired by nutrient deprivation or depletion, transcription of rRNA genes declines, and this decline is reversed when growth-permissive conditions are restored. Because growth-rate dependence is a universal feature of rRNA gene regulation, determining the molecular mechanisms coupling pol I activity to cell growth is a central question in studies of both yeast and mammalian systems.Transcription of rRNA genes of Saccharomyces cerevisiae requires the activity of at least three transcription factors that have been both genetically and biochemically defined. Two of these are multisubunit factors that interact directly with distinct elements of the rDNA promoter to assemble a preinitiation complex. Core factor is composed of three essential gene products, RRN6, RRN7, and RRN11, associates with TATAbinding protein, and is required to direct initiation from the core promoter in vitro and in vivo (1-5). Upstream activation factor (UAF) binds to the upstream element and stimulates transcription from the core promoter. When the genes encoding the UAF subunits RRN5, RRN9, or RRN10 are individually disrupted, cells remain viable but exhibit pronounced growth defects, indicating that UAF activity is necessary to support levels of rRNA synthesis required for normal cell growth (6). UAF subunits interact with core factor subunits in vitro, and direct interaction of UAF with TATA-binding protein has been shown to mediate transcriptional activation in vivo (3, 7). The third transcription factor, Rrn3, is unique in that it functions as a single subunit, shows no sequence-specific DNA-binding activity, and is not required for preinitiation complex assembly (8). Rrn3 appears instead to function by direct interaction with RNA polymerase because it is stably associated with pol I in transcriptionally active extracts (9), and its transcription activity is enhanced by preincubation with pol I in the absence of either DNA template or other pol I transcription factors (4,8). Interestingly, the interaction of Rrn3 ...

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“…In vitro data suggests that Syc1p/Rrn3p may serve to "activate" pol I (56), and it is possible that Cbf5p functions upstream of Syc1p/ Rrn3p in an rRNA transcriptional-control pathway. The availability of a well-defined in vitro pol I-dependent transcription system in yeast (46,56) should eventually lead to a definition of the relationship between these proteins, as well as of the molecular role of Cbf5p in rRNA synthesis.…”

Section: Discussionmentioning

confidence: 99%

The Yeast Nucleolar Protein Cbf5p Is Involved in rRNA Biosynthesis and Interacts Genetically with the RNA Polymerase I Transcription Factor RRN3

Cadwell

Yoon

Zebarjadian

et al. 1997

Molecular and Cellular Biology

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Carbon, Mol. Cell. Biol. 13:4884-4893, 1993). Cbf5p also binds microtubules in vitro and interacts genetically with two known centromere-related protein genes (NDC10/CBF2 and MCK1). However, Cbf5p was found to be nucleolar and is highly homologous to the rat nucleolar protein NAP57, which coimmunoprecipitates with Nopp140 and which is postulated to be involved in nucleolarcytoplasmic shuttling (U. T. Meier, and G. Blobel, J. Cell Biol. 127:1505-1514, 1994). The temperaturesensitive cbf5-1 mutant demonstrates a pronounced defect in rRNA biosynthesis at restrictive temperatures, while tRNA transcription and pre-rRNA and pre-tRNA cleavage processing appear normal. The cbf5-1 mutant cells are deficient in cytoplasmic ribosomal subunits at both permissive and restrictive temperatures. A high-copy-number yeast genomic library was screened for genes that suppress the cbf5-1 temperature-sensitive growth phenotype. SYC1 (suppressor of yeast cbf5-1) was identified as a multicopy suppressor of cbf5-1 and subsequently was found to be identical to RRN3, an RNA polymerase I transcription factor. A cbf5⌬ null mutant is not rescued by plasmid pNOY103 containing a yeast 35S rRNA gene under the control of a Pol II promoter, indicating that Cbf5p has one or more essential functions in addition to its role in rRNA transcription.Cbf5p of the yeast Saccharomyces cerevisiae was originally isolated as one of the major low-affinity centromeric DNA (CEN) binding proteins (23). CBF5 is an essential gene encoding a highly charged protein with a domain containing ten tandem KKE/D repeats. These repeats are homologous to portions of microtubule-associated proteins 1A and 1B in the domain responsible for microtubule binding (40), and Cbf5p has been shown to bind microtubules in vitro (23). Yeast cells containing C-terminal truncated CBF5 genes delay, with replicated genomes, at the G 2 /M phase of the cell cycle, with the replicated DNA being located at the bud junction (23). Overexpression of Cbf5p suppresses the ndc10-1 temperature-sensitive (ts) mutation in the gene specifying the 110-kDa subunit (Cbf2p/Ndc10p) of the multisubunit yeast centromere DNA binding complex, CBF3, and overexpression of meiosis and centromere regulatory kinase Mck1p suppresses a ts mutation in the gene for either Cbf2p or Cbf5p (22). These genetic interactions support a direct or indirect link between Cbf5p and centromeres. However, both Cbf5p and a homologous protein (NAP57) from rats were found to be nucleolar proteins (21,33). While a functional relationship between a nucleolar protein and centromeres is not obvious, some proteins are associated with both the nucleolus and the centromere in higher eukaryotes. It has been known for some time that centromere autoantigens associate with the nucleolus (43), and experiments with autoimmune sera have also shown that a set of nucleolar proteins and ribonuclear proteins relocate around chromosomes during mitosis (16,17).The nucleolar location of Cbf5p may be indicative of a function unrelated to centromeres and chromos...

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Characterization of the Components of Reconstituted Saccharomyces cerevisiae RNA Polymerase I Transcription Complexes

Cited by 21 publications

References 36 publications

Regulation of the RNA Polymerase I and III Transcription Systems in Response to Growth Conditions

Regulation of the RNA Polymerase I and III Transcription Systems in Response to Growth Conditions

RNA polymerase I transcription factor Rrn3 is functionally conserved between yeast and human

The Yeast Nucleolar Protein Cbf5p Is Involved in rRNA Biosynthesis and Interacts Genetically with the RNA Polymerase I Transcription Factor RRN3

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