Mitochondrial and nuclear mitoribosomal suppressors that enable misreading of ochre codons in yeast mitochondria

Kruszewska, Anna; Słonimski, Piotr P.

doi:10.1007/bf00396198

Cited by 30 publications

(15 citation statements)

References 16 publications

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“…Of 334 mutations tested, 16 were suppressed by NAM9-1. Its action spectrum resembled that of the nam3 suppressor (37). Like the latter suppressor, NAM9 suppressed some mutations in the nonsplit oxil and oxi2 genes, as well as mutations in the mosaic genes oxi3 and cob-box.…”

Section: Resultsmentioning

confidence: 93%

“…NAM], NAM2, NAM7, and NAM8 suppressors are involved in both translation and splicing (2,13,26,40). The recessive suppressor mutations nam3 and ribosome series suppressors (5,36,37,68) seem to be analogous to Escherichia coli ribosomal ram suppressors in the sense of their allelespecific, gene-nonspecific mode of action (18). Those suppressors presumably act by decreasing the fidelity of translation resulting from the changes in mitochondrial r-proteins.…”

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confidence: 99%

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NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

Boguta¹,

Dmochowska²,

Borsuk³

et al. 1992

Mol. Cell. Biol.

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We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxil gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxil-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rhoo strain.Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation.Mitochondria possess their own translation apparatus responsible for the synthesis of only a handful of the hundreds of mitochondrial proteins (for a review, see reference 59a). The biogenesis of this apparatus depends on the coordinate expression of both mitochondrial and nuclear genes (9). Although the whole set of tRNAs required for mitochondrial translation and the rRNAs of mitochondrial ribosomes are encoded by the mitochondrial genes, the mitochondrial ribosomal proteins, as well as other elements of the mitochondrial translation system, are encoded by nuclear genes and transported to mitochondria. The proteins of mitochondrial ribosomes differ from those of cytoplasmic ribosomes. Thus, two different sets of nuclear genes code for mitochondrial and cytoplasmic ribosomal proteins (r-proteins). During the last few years, genes for approximately half of the 70 to 80 yeast cytoplasmic r-proteins have been isolated and characterized (for a review, see reference 52). At present, however, very little is known about the structure and organization of genes for the mitochondrial r-proteins (MRP genes). So far, sequences for only 10 of 60 to 70 genes for mitochondrial r-proteins have been published (10, 10a, 14, 23, 33, 45-46, 51, 59). Thus, to gain more insight into the structure, chromosomal organization, and evolution of MRP genes, a more comprehe...

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Section: Resultsmentioning

confidence: 93%

mentioning

confidence: 99%

NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

Boguta¹,

Dmochowska²,

Borsuk³

et al. 1992

Mol. Cell. Biol.

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“…The wild type strain W301-1B with the URA3 marker integrated into the chromosomal MRF1 locus was crossed with R13 and several other suppressors, including the CK311/B145 strain carrying the nam3-1 mutation (27). Diploids were sporulated, and Ͼ10 tetrads from each cross were analyzed for segregation of Ura ϩ and suppressor phenotypes.…”

Section: Identification Of Mit ϫ Suppressors As Mutations In the Mrf1mentioning

confidence: 99%

Mutations in the Yeast MRF1 Gene Encoding Mitochondrial Release Factor Inhibit Translation on Mitochondrial Ribosomes

Towpik

Chacińska

Cieśla

et al. 2004

Journal of Biological Chemistry

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Although the control of mitochondrial translation in the yeast Saccharomyces cerevisiae has been studied extensively, the mechanism of termination remains obscure. Ten mutations isolated in a genetic screen for read-through of premature stop codons in mitochondrial genes were localized in the chromosomal gene encoding the mitochondrial release factor mRF1. The mrf1-13 and mrf1-780 mutant genes, in contrast to other alleles, caused a non-respiratory phenotype that correlated with decreased expression of mitochondrial genes as well as a reporter ARG8 m gene inserted into mitochondrial DNA. The steady-state levels of several mitochondrially encoded proteins, but not their mRNAs, were dramatically decreased in mrf1-13 and mrf1-780 cells. Structural models of mRF1 were constructed, allowing localization of residues substituted in the mrf1 mutants and offering an insight into the possible mechanism by which these mutations change the mitochondrial translation termination fidelity. Inhibition of mitochondrial translation in mrf1-13 and mrf1-780 correlated with the three-dimensional localization of the mutated residues close to the PST motif presumably involved in the recognition of stop codons in mitochondrial mRNA.The mitochondrial genetic system of the yeast Saccharomyces cerevisiae has been a subject of intensive studies. Seven genes in the yeast mitochondrial DNA (mtDNA) 1 encode integral membrane proteins that are assembled together with subunits encoded in the nuclear DNA into multimeric respiratory complexes. Maintenance and expression of mtDNA require approximately 200 proteins encoded in the nuclear DNA and imported to mitochondria from the cytosol (1, 2). Among them are mitochondrial ribosomal proteins, general translation factors, and translational activators. Current analysis of the proteome of yeast mitochondria allowed for identification of 65 mitoribosomal proteins (2); however, only ϳ60% of these have recognizable homology with bacterial ribosomal proteins (3). Multiple mitoribosome-specific proteins in yeast and mammals demonstrate the considerable divergence of mitochondrial ribosomes from their prokaryotic ancestors (4, 5). The mechanisms that control mitochondrial translation are difficult to study because no in vitro system for the expression of translation products encoded in mtDNA has yet been established. In contrast to the biochemical difficulties, the yeast S. cerevisiae, being a facultative anaerobe, offers distinct advantages for mitochondrial genetic studies. Several hundred point mutations or small deletions called mit Ϫ have been genetically mapped and assigned to genes in mtDNA. We have long been involved in a search for suppressors of mit Ϫ mutants that act at the level of mitochondrial translation. Analogous suppressor approach applied to chromosomal mutants allowed identification of proteins responsible for translation fidelity in the yeast cytosolic system. Ribosomal proteins from the decoding center of the ribosome and translation termination factors represented two classes of prote...

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“…On the other hand, the situation is ideal in yeast mitochondria where the exceptional existence of only one copy of the small and large rRNA genes should allow the isolation of rRNA suppressors, if not lethal. Indeed, such suppressors have been isolated already: mim3-1 by 56 and MSU1 by 7.…”

Section: Introductionmentioning

confidence: 99%

A single mutation in the 15S rRNA gene confers non sense suppressor activity and interacts with mRF1 the release factor in yeast mitochondria

Gargouri¹,

Macadré²,

Lazowska³

2015

MIC

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We have determined the nucleotide sequence of the mim3-1 mitochondrial ribosomal suppressor, acting on ochre mitochondrial mutations and one frameshift mutation in Saccharomyces cerevisiae. The 15s rRNA suppressor gene contains a G633 to C transversion. Yeast mitochondrial G633 corresponds to G517 of the E.coli 15S rRNA, which is occupied by an invariant G in all known small rRNA sequences. Interestingly, this mutation has occurred at the same position as the known MSU1 mitochondrial suppressor which changes G633 to A. The suppressor mutation lies in a highly conserved region of the rRNA, known in E.coli as the 530-loop, interacting with the S4, S5 and S12 ribosomal proteins. We also show an interesting interaction between the mitochondrial mim3-1 and the nuclear nam3-1 suppressors, both of which have the same action spectrum on mitochondrial mutations: nam3-1 abolishes the suppressor effect when present with mim3-1 in the same haploid cell. We discuss these results in the light of the nature of Nam3, identified by 1 as the yeast mitochondrial translation release factor. A hypothetical mechanism of suppression by "ribosome shifting" is also discussed in view of the nature of mutations suppressed and not suppressed.

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Mitochondrial and nuclear mitoribosomal suppressors that enable misreading of ochre codons in yeast mitochondria

Cited by 30 publications

References 16 publications

NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

Mutations in the Yeast MRF1 Gene Encoding Mitochondrial Release Factor Inhibit Translation on Mitochondrial Ribosomes

A single mutation in the 15S rRNA gene confers non sense suppressor activity and interacts with mRF1 the release factor in yeast mitochondria

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