Saccharomyces cerevisiae Sof1p Associates with 35S Pre-rRNA Independent from U3 snoRNA and Rrp5p

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene. Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu). Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced. The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro. Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts. A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.

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Defective processing of ribosomal precursor RNA in Saccharomyces cerevisiae

Biochemical Journal ◽

10.1042/bj2200461 ◽

1984 ◽

Vol 220 (2) ◽

pp. 461-467 ◽

Cited By ~ 4

Author(s):

J A Mitlin ◽

M Cannon

Keyword(s):

Saccharomyces Cerevisiae ◽

Yeast Strain ◽

Ribosome Biogenesis ◽

Sucrose Gradient ◽

Gradient Analysis ◽

Ribosomal Subunits ◽

Abnormal Distribution ◽

Precursor Rna ◽

Ribosomal Precursor

Saccharomyces cerevisiae (strain A224A) has an abnormal distribution of cytoplasmic ribosomal subunits when grown at 36 degrees C, with sucrose-gradient analysis of extracts revealing an apparent excess of material sedimenting at 60 S. This abnormality is not observed at either 23 degrees C or 30 degrees C. At 36 degrees C the defect(s) is expressed as a slowed conversion of 20 S ribosomal precursor RNA to mature 18 S rRNA, although the corresponding maturation of 27 S ribosomal precursor RNA to mature 25 S rRNA is normal. Studies on this yeast strain and on mutants derived from it may help to elucidate the role(s) of individual ribosomal components in controlling ribosome biogenesis in eukaryotes.

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Ribosome association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3027 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3027-3036 ◽

Cited By ~ 100

Author(s):

M Ramirez ◽

R C Wek ◽

A G Hinnebusch

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Protein Kinase ◽

Protein Accumulation ◽

Yeast Saccharomyces Cerevisiae ◽

Ribosomal Subunits ◽

Cell Extracts ◽

Ribosome Association

The GCN4 gene of the yeast Saccharomyces cerevisiae encodes a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level according to the availability of amino acids. GCN2 is a protein kinase required for increased translation of GCN4 mRNA in amino acid-starved cells. Centrifugation of cell extracts in sucrose gradients indicated that GCN2 comigrates with ribosomal subunits and polysomes. The fraction of GCN2 cosedimenting with polysomes was reduced under conditions in which polysomes were dissociated, suggesting that GCN2 is physically bound to these structures. When the association of 40S and 60S subunits was prevented by omitting Mg2+ from the gradient, almost all of the GCN2 comigrated with 60S ribosomal subunits, and it remained bound to these particles during gel electrophoresis under nondenaturing conditions. GCN2 could be dissociated from 60S subunits by 0.5 M KCl, suggesting that it is loosely associated with ribosomes rather than being an integral ribosomal protein. Accumulation of GCN2 on free 43S-48S particles and 60S subunits occurred during polysome runoff in vitro and under conditions of reduced growth rate in vivo. These observations, plus the fact that GCN2 shows preferential association with free ribosomal subunits during exponential growth, suggest that GCN2 interacts with ribosomes during the translation initiation cycle. The extreme carboxyl-terminal segment of GCN2 is essential for its interaction with ribosomes. These sequences are also required for the ability of GCN2 to stimulate GCN4 translation in vivo, leading us to propose that ribosome association by GCN2 is important for its access to substrates in the translational machinery or for detecting uncharged tRNA in amino acid-starved cells.

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Ribosome association of GCN2 protein kinase, a translational activator of the GCN4 gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.6.3027-3036.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 3027-3036 ◽

Cited By ~ 1

Author(s):

M Ramirez ◽

R C Wek ◽

A G Hinnebusch

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Protein Kinase ◽

Protein Accumulation ◽

Yeast Saccharomyces Cerevisiae ◽

Ribosomal Subunits ◽

Cell Extracts ◽

Ribosome Association

The GCN4 gene of the yeast Saccharomyces cerevisiae encodes a transcriptional activator of amino acid biosynthetic genes that is regulated at the translational level according to the availability of amino acids. GCN2 is a protein kinase required for increased translation of GCN4 mRNA in amino acid-starved cells. Centrifugation of cell extracts in sucrose gradients indicated that GCN2 comigrates with ribosomal subunits and polysomes. The fraction of GCN2 cosedimenting with polysomes was reduced under conditions in which polysomes were dissociated, suggesting that GCN2 is physically bound to these structures. When the association of 40S and 60S subunits was prevented by omitting Mg2+ from the gradient, almost all of the GCN2 comigrated with 60S ribosomal subunits, and it remained bound to these particles during gel electrophoresis under nondenaturing conditions. GCN2 could be dissociated from 60S subunits by 0.5 M KCl, suggesting that it is loosely associated with ribosomes rather than being an integral ribosomal protein. Accumulation of GCN2 on free 43S-48S particles and 60S subunits occurred during polysome runoff in vitro and under conditions of reduced growth rate in vivo. These observations, plus the fact that GCN2 shows preferential association with free ribosomal subunits during exponential growth, suggest that GCN2 interacts with ribosomes during the translation initiation cycle. The extreme carboxyl-terminal segment of GCN2 is essential for its interaction with ribosomes. These sequences are also required for the ability of GCN2 to stimulate GCN4 translation in vivo, leading us to propose that ribosome association by GCN2 is important for its access to substrates in the translational machinery or for detecting uncharged tRNA in amino acid-starved cells.

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Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.8.1.361 ◽

1988 ◽

Vol 8 (1) ◽

pp. 361-370 ◽

Cited By ~ 2

Author(s):

S Ganguly ◽

P A Sharp ◽

U L RajBhandary

Keyword(s):

Saccharomyces Cerevisiae ◽

Mammalian Cells ◽

Wild Type ◽

Cell Extracts ◽

Amber Suppressor ◽

Gene Transcripts ◽

A Site ◽

Made In

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene. Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu). Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced. The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro. Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts. A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.

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PENGARUH KONSENTRASI RAGI Saccharomyces cerevisiae DAN LAMA FERMENTASI TERHADAP KUALITAS CUKA TOMI-TOMI (Flacourtia inermis)

AGRITEKNO Jurnal Teknologi Pertanian ◽

10.30598/jagritekno.2015.4.2.50 ◽

2015 ◽

Vol 4 (2) ◽

pp. 50-55

Author(s):

Sandra J Nendissa ◽

Rachel Breemer ◽

Nikholaus Melamas

Keyword(s):

Saccharomyces Cerevisiae ◽

Experimental Design ◽

Total Dissolved Solids ◽

Total Sugar ◽

Dissolved Solids ◽

Randomized Experimental Design ◽

Two Factors ◽

Four Levels

This objectives of this research were both to study and determine the best level of concentration of yeast Saccharomyces cereviseae and period of fermentation on the quality of tomi-tomi vinegar (Flacourtia inermis). A completely randomized experimental design with two factors of treatment was applied in this research. The first factor was concentration of yeast S. cereviseae having four levels of tretament, i.e.: without the addition of yeast 0.5, 1 and 1.5 g yeast. The second factor was period fermentation with 1, 2, 3, 4, and 5 weeks. The result indicated that the concentration of yeast S. cereviseae 1.5 g and period fermentation 5 week produced a good tomi-tomi vinegar with total acids 51.22%, total dissolved solids 8.35, total sugar 8.07% and pH 5.40.

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Requirement for Three Novel Protein Complexes in the Absence of the Sgs1 DNA Helicase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.1.103 ◽

2001 ◽

Vol 157 (1) ◽

pp. 103-118 ◽

Cited By ~ 16

Author(s):

Janet R Mullen ◽

Vivek Kaliraman ◽

Samer S Ibrahim ◽

Steven J Brill

Keyword(s):

Saccharomyces Cerevisiae ◽

Genome Stability ◽

Protein Complexes ◽

Ring Finger ◽

Dna Helicase ◽

Open Reading Frames ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dna Helicases ◽

Cell Extracts

Abstract The Saccharomyces cerevisiae Sgs1 protein is a member of the RecQ family of DNA helicases and is required for genome stability, but not cell viability. To identify proteins that function in the absence of Sgs1, a synthetic-lethal screen was performed. We obtained mutations in six complementation groups that we refer to as SLX genes. Most of the SLX genes encode uncharacterized open reading frames that are conserved in other species. None of these genes is required for viability and all SLX null mutations are synthetically lethal with mutations in TOP3, encoding the SGS1-interacting DNA topoisomerase. Analysis of the null mutants identified a pair of genes in each of three phenotypic classes. Mutations in MMS4 (SLX2) and SLX3 generate identical phenotypes, including weak UV and strong MMS hypersensitivity, complete loss of sporulation, and synthetic growth defects with mutations in TOP1. Mms4 and Slx3 proteins coimmunoprecipitate from cell extracts, suggesting that they function in a complex. Mutations in SLX5 and SLX8 generate hydroxyurea sensitivity, reduced sporulation efficiency, and a slow-growth phenotype characterized by heterogeneous colony morphology. The Slx5 and Slx8 proteins contain RING finger domains and coimmunoprecipitate from cell extracts. The SLX1 and SLX4 genes are required for viability in the presence of an sgs1 temperature-sensitive allele at the restrictive temperature and Slx1 and Slx4 proteins are similarly associated in cell extracts. We propose that the MMS4/SLX3, SLX5/8, and SLX1/4 gene pairs encode heterodimeric complexes and speculate that these complexes are required to resolve recombination intermediates that arise in response to DNA damage, during meiosis, and in the absence of SGS1/TOP3.

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The Putative RNA Helicase Dbp6p Functionally Interacts With Rpl3p, Nop8p and the Novel trans-acting Factor Rsa3p During Biogenesis of 60S Ribosomal Subunits in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/166.4.1687 ◽

2004 ◽

Vol 166 (4) ◽

pp. 1687-1699

Author(s):

Jesús de la Cruz ◽

Thierry Lacombe ◽

Olivier Deloche ◽

Patrick Linder ◽

Dieter Kressler

Keyword(s):

Saccharomyces Cerevisiae ◽

Ribosome Biogenesis ◽

Null Allele ◽

Ribosomal Subunit ◽

Interaction Network ◽

The Novel ◽

Rna Helicases ◽

Ribosomal Subunits ◽

Synthetic Lethal ◽

Synthetically Lethal

Abstract Ribosome biogenesis requires at least 18 putative ATP-dependent RNA helicases in Saccharomyces cerevisiae. To explore the functional environment of one of these putative RNA helicases, Dbp6p, we have performed a synthetic lethal screen with dbp6 alleles. We have previously characterized the nonessential Rsa1p, whose null allele is synthetically lethal with dbp6 alleles. Here, we report on the characterization of the four remaining synthetic lethal mutants, which reveals that Dbp6p also functionally interacts with Rpl3p, Nop8p, and the so-far-uncharacterized Rsa3p (ribosome assembly 3). The nonessential Rsa3p is a predominantly nucleolar protein required for optimal biogenesis of 60S ribosomal subunits. Both Dbp6p and Rsa3p are associated with complexes that most likely correspond to early pre-60S ribosomal particles. Moreover, Rsa3p is co-immunoprecipitated with protA-tagged Dbp6p under low salt conditions. In addition, we have established a synthetic interaction network among factors involved in different aspects of 60S-ribosomal-subunit biogenesis. This extensive genetic analysis reveals that the rsa3 null mutant displays some specificity by being synthetically lethal with dbp6 alleles and by showing some synthetic enhancement with the nop8-101 and the rsa1 null allele.

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