Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae

1985 ◽  
Vol 5 (12) ◽  
pp. 3429-3435
Author(s):  
N Abovich ◽  
L Gritz ◽  
L Tung ◽  
M Rosbash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Ribosomal Protein ◽  
Dosage Compensation ◽  
Ribosomal Proteins ◽  
Gene Dosage ◽  
Translational Efficiency ◽  
Phenotypic Analysis ◽  
Ribosomal Subunits ◽  
Single Deletion

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.

scholarly journals Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

1985 ◽  
Vol 5 (12) ◽  
pp. 3429-3435 ◽  
Author(s):  
N Abovich ◽  
L Gritz ◽  
L Tung ◽  
M Rosbash
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Growth Rate ◽  
Ribosomal Protein ◽  
Dosage Compensation ◽  
Ribosomal Proteins ◽  
Gene Dosage ◽  
Translational Efficiency ◽  
Phenotypic Analysis ◽  
Ribosomal Subunits ◽  
Single Deletion

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.

scholarly journals Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (10) ◽  
pp. 5235-5243 ◽  
Author(s):  
D M Baronas-Lowell ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Wild Type ◽  
Diploid Strain ◽  
Chain Reaction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Subunits ◽  
Strong Homology ◽  
Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

Ribosomal protein L30 is dispensable in the yeast Saccharomyces cerevisiae

1990 ◽  
Vol 10 (10) ◽  
pp. 5235-5243
Author(s):  
D M Baronas-Lowell ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Wild Type ◽  
Diploid Strain ◽  
Chain Reaction ◽  
Yeast Saccharomyces Cerevisiae ◽  
Ribosomal Subunits ◽  
Strong Homology ◽  
Polymerase Chain

In the yeast Saccharomyces cerevisiae, L30 is one of many ribosomal proteins that is encoded by two functional genes. We have cloned and sequenced RPL30B, which shows strong homology to RPL30A. Use of mRNA as a template for a polymerase chain reaction demonstrated that RPL30B contains an intron in its 5' untranslated region. This intron has an unusual 5' splice site, C/GUAUGU. The genomic copies of RPL30A and RPL30B were disrupted by homologous recombination. Growth rates, primer extension, and two-dimensional ribosomal protein analyses of these disruption mutants suggested that RPL30A is responsible for the majority of L30 production. Surprisingly, meiosis of a diploid strain carrying one disrupted RPL30A and one disrupted RPL30B yielded four viable spores. Ribosomes from haploid cells carrying both disrupted genes had no detectable L30, yet such cells grew with a doubling time only 30% longer than that of wild-type cells. Furthermore, depletion of L30 did not alter the ratio of 60S to 40S ribosomal subunits, suggesting that there is no serious effect on the assembly of 60S subunits. Polysome profiles, however, suggest that the absence of L30 leads to the formation of stalled translation initiation complexes.

scholarly journals Hybrid Protein between Ribosomal Protein S16 and RimM of Escherichia coli Retains the Ribosome Maturation Function of Both Proteins

2001 ◽  
Vol 183 (18) ◽  
pp. 5352-5357 ◽  
Author(s):  
J. Mattias Lövgren ◽  
P. Mikael Wikström
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Ribosomal Protein ◽  
Ribosome Biogenesis ◽  
Stop Codon ◽  
Translational Efficiency ◽  
Hybrid Protein ◽  
Ribosomal Subunits ◽  
Suppressor Mutations ◽  
Substitution Mutant

ABSTRACT The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of E. coli approximately threefold. Several faster-growing derivatives of the rimM amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the rimMmRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the rimM mRNA. Three other independently isolated suppressor mutations created a fusion betweenrpsP, encoding the ribosomal protein S16, andrimM on the chromosome as a result of mutations in therpsP stop codon preceding rimM. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.

scholarly journals Effects of progressive depletion of TCM1 or CYH2 mRNA on Saccharomyces cerevisiae ribosomal protein accumulation.

1986 ◽  
Vol 6 (5) ◽  
pp. 1535-1544 ◽  
Author(s):  
H G Nam ◽  
H M Fried
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Normal Level ◽  
Yeast Strain ◽  
Ribosomal Proteins ◽  
Translational Efficiency ◽  
Protein Accumulation ◽  
Subunit Assembly ◽  
Cell Biol

When present in excess, the mRNAs for Saccharomyces cerevisiae ribosomal proteins L3 and L29 are translated less efficiently, so that synthesis of these proteins remains commensurate with that of other ribosomal proteins (N.J. Pearson, H.M. Fried, and J.R. Warner, Cell 29:347-355, 1982; J.R. Warner, G. Mitra, W.F. Schwindinger, M. Studeny, and H.M. Fried, Mol. Cell. Biol. 5:1512-1521, 1985). We used a yeast strain with a conditionally transcribed derivative of the L3 gene to deplete cells progressively of L3 mRNA. In this case translation of L3 mRNA did not become more efficient so that L3 was not maintained at a normal level. Even when there was an initial excess of L3 mRNA, interruption of its further transcription produced an immediate drop in L3 synthesis, suggesting that the translational efficiency of preexisting mRNA cannot be altered. Lack of L3 synthesis afforded an opportunity to examine coordinate accumulation of other ribosomal proteins. Without L3, apparent synthesis of several 60S subunit proteins diminished, and 60S subunits did not assemble. A similar phenomenon occurred when, in a second strain, synthesis of ribosomal protein L29 was prevented. Loss of 60S subunit assembly was accompanied by a destabilization of some 60S ribosomal protein mRNAs. These data suggest that synthesis of some S. cerevisiae ribosomal proteins may be regulated posttranscriptionally as a function of the extent to which they are assembled.

scholarly journals Effects of progressive depletion of TCM1 or CYH2 mRNA on Saccharomyces cerevisiae ribosomal protein accumulation

1986 ◽  
Vol 6 (5) ◽  
pp. 1535-1544
Author(s):  
H G Nam ◽  
H M Fried
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Normal Level ◽  
Yeast Strain ◽  
Ribosomal Proteins ◽  
Translational Efficiency ◽  
Protein Accumulation ◽  
Subunit Assembly ◽  
Cell Biol

When present in excess, the mRNAs for Saccharomyces cerevisiae ribosomal proteins L3 and L29 are translated less efficiently, so that synthesis of these proteins remains commensurate with that of other ribosomal proteins (N.J. Pearson, H.M. Fried, and J.R. Warner, Cell 29:347-355, 1982; J.R. Warner, G. Mitra, W.F. Schwindinger, M. Studeny, and H.M. Fried, Mol. Cell. Biol. 5:1512-1521, 1985). We used a yeast strain with a conditionally transcribed derivative of the L3 gene to deplete cells progressively of L3 mRNA. In this case translation of L3 mRNA did not become more efficient so that L3 was not maintained at a normal level. Even when there was an initial excess of L3 mRNA, interruption of its further transcription produced an immediate drop in L3 synthesis, suggesting that the translational efficiency of preexisting mRNA cannot be altered. Lack of L3 synthesis afforded an opportunity to examine coordinate accumulation of other ribosomal proteins. Without L3, apparent synthesis of several 60S subunit proteins diminished, and 60S subunits did not assemble. A similar phenomenon occurred when, in a second strain, synthesis of ribosomal protein L29 was prevented. Loss of 60S subunit assembly was accompanied by a destabilization of some 60S ribosomal protein mRNAs. These data suggest that synthesis of some S. cerevisiae ribosomal proteins may be regulated posttranscriptionally as a function of the extent to which they are assembled.

Mild temperature shock alters the transcription of a discrete class of Saccharomyces cerevisiae genes

1983 ◽  
Vol 3 (3) ◽  
pp. 457-465
Author(s):  
C H Kim ◽  
J R Warner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Temperature Shift ◽  
Restrictive Temperature ◽  
Ribosomal Protein Genes ◽  
Wild Type ◽  
Temperature Shock ◽  
Mild Temperature ◽  
Mutant Cells

In Saccharomyces cerevisiae the synthesis of ribosomal proteins declines temporarily after a culture has been subjected to a mild temperature shock, i.e., a shift from 23 to 36 degrees C, each of which support growth. Using cloned genes for several S. cerevisiae ribosomal proteins, we found that the changes in the synthesis of ribosomal proteins parallel the changes in the concentration of mRNA of each. The disappearance and reappearance of the mRNA is due to a brief but severe inhibition of the transcription of each of the ribosomal protein genes, although the total transcription of mRNA in the cells is relatively unaffected by the temperature shock. The precisely coordinated response of these genes, which are scattered throughout the genome, suggests that either they or the enzyme which transcribes them has unique properties. In certain S. cerevisiae mutants, the synthesis of ribosomal proteins never recovers from a temperature shift. Yet both the decline and the resumption of transcription of these genes during the 30 min after the temperature shift are indistinguishable from those in wild-type cells. The failure of the mutant cells to grow at the restrictive temperature appears to be due to their inability to process the RNA transcribed from genes which have introns (Rosbash et al., Cell 24:679-686, 1981), a large proportion of which appear to be ribosomal protein genes.

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells

1983 ◽  
Vol 3 (2) ◽  
pp. 190-197
Author(s):  
J J Madjar ◽  
M Frahm ◽  
S McGill ◽  
D J Roufa
Keyword(s):  
Ribosomal Protein ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Chinese Hamster ◽  
Complementation Group ◽  
Resistance Mutations ◽  
Cho Cell ◽  
Ribosomal Subunits ◽  
40S Ribosomal Subunit ◽  
One Step

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.

scholarly journals Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family.

Genetics ◽  
1993 ◽  
Vol 135 (3) ◽  
pp. 719-730
Author(s):  
A G Paulovich ◽  
J R Thompson ◽  
J C Larkin ◽  
Z Li ◽  
J L Woolford
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Gene Fusion ◽  
Null Allele ◽  
Ribosomal Subunit ◽  
Null Alleles ◽  
Protein Synthesis Inhibitor ◽  
Lacz Gene ◽  
Ribosomal Subunits ◽  
Cry1 Gene

Abstract The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype. (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.

scholarly journals Gene dosage alteration of L2 ribosomal protein genes in Saccharomyces cerevisiae: effects on ribosome synthesis.

1988 ◽  
Vol 8 (11) ◽  
pp. 4792-4798 ◽  
Author(s):  
A Lucioli ◽  
C Presutti ◽  
S Ciafrè ◽  
E Caffarelli ◽  
P Fragapane ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Protein ◽  
Gene Dosage ◽  
Transcriptional Level ◽  
Ribosomal Protein Genes ◽  
Promoter Regions ◽  
Phenotypic Alteration ◽  
Mrna Pool ◽  
Gene Copies ◽  
The Difference

In Saccharomyces cerevisiae, the genes coding for the ribosomal protein L2 are present in two copies per haploid genome. The two copies, which encode proteins differing in only a few amino acids, contribute unequally to the L2 mRNA pool: the L2A copy makes 72% of the mRNA, while the L2B copy makes only 28%. Disruption of the L2B gene (delta B strain) did not lead to any phenotypic alteration, whereas the inactivation of the L2A copy (delta A strain) produced a slow-growth phenotype associated with decreased accumulation of 60S subunits and ribosomes. No intergenic compensation occurred at the transcriptional level in the disrupted strains; in fact, delta A strains contained reduced levels of L2 mRNA, whereas delta B strains had almost normal levels. The wild-type phenotype was restored in the delta A strains by transformation with extra copies of the intact L2A or L2B gene. As already shown for other duplicated genes (Kim and Warner, J. Mol. Biol. 165:79-89, 1983; Leeret al., Curr. Genet. 9:273-277, 1985), the difference in expression of the two gene copies could be accounted for via differential transcription activity. Sequence comparison of the rpL2 promoter regions has shown the presence of canonical HOMOL1 boxes which are slightly different in the two genes.

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