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Structure, function and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP13, a protein of the small subunit of the mitochondrial ribosome
Abstract
MRP13 was defined by biochemical criteria as a 35-kilodalton small subunit protein of the yeast mitochondrial ribosome. The MRP13 gene was identified by immunological screening of a yeast genomic library in $\lambda$gt11 and a functional copy of the gene has been cloned on a 2.2-kilobase BglII fragment. Sequencing of this fragment showed that the MRP13 coding region specifies as 324-amino acid basic protein with a calculated M$\sb{\rm r}$ of 37,366. Computer searches failed to reveal any significant sequence similarity to previously identified ribosomal proteins or to the sequences in the current National Biomedical Research Foundation data base. Cells carrying disrupted copies of MRP13 lacked the MRP13 protein but were not impaired for growth on nonfermentable carbon sources. However, in comparison to the wild type, mrp13-$\Delta$2::TRP1 mutant cells had a lower rate of whole cell respiration, an unusual profile on in vivo labeled mitochondrial translation products and an abnormal profile of ribosomal subunits in sucrose gradient centrifugation. Mutants lacking MRP13 were also impaired in their ability to undergo the transition from growth on high concentrations of glucose to growth on nonrepressing carbon sources. This mutant phenotype suggests an important role for the MRP13 protein under conditions where cells are actively increasing their capacity for the synthesis of mitochondrial encoded proteins. Analysis of the sequence in the MRP13 5$\sp\prime$-flanking region revealed the closely linked gene for the cytoplasmic ribosomal protein RP39A. The RP39A coding region begins at nucleotide $-$846 and ends at $-$325 with respect to the MRP13 translational start. The steady-state levels of the MRP13 mRNA were determined in response to carbon catabolite repression, variation in the mitochondrial genetic background, and increased gene dosage of MRP13. In $\rho\sp+$ cells, transcript levels were repressed severalfold by growth in glucose as compared with growth in either galactose or nonfermentable carbon sources. In respiratory-deficient strains ($\rho\sp{\rm o,}$ mit$\sp-$), however, transcription appeared to be largely derepressed even in the presence of high concentrations of glucose. Thus, the MRP13 mRNA is a member of a class of yeast nucleus-encoded RNAs whose transcription responds to changes in the mitochondrial genetic background. Despite high levels of the MRP13 transcripts in $\rho\sp{\rm o}$ cells, the MRP13 protein did not accumulate, suggesting that the protein is relatively unstable in the absence of ribosome assembly. Cells carrying the MRP13 gene on a multiple-copy plasmid overproduced the mRNA in rough proportion to the gene dosage and the protein accumulated to a significant but lesser extent. The results indicate that MRP13 expression is regulated predominantly at the transcriptional level in response to catabolite repression and the cellular capacity for respiration. In addition, the levels of the MRP13 protein appear to be modulated posttranscriptionally by degradation of excess, unassembled polypeptides.
Subject Area
Molecular biology
Recommended Citation
Partaledis, Judith A, "Structure, function and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP13, a protein of the small subunit of the mitochondrial ribosome" (1990). Doctoral Dissertations Available from Proquest. AAI9022731.
https://scholarworks.umass.edu/dissertations/AAI9022731