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

Abstract

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...

The Yeast Translation Release Factors Mrf1p and Sup45p (eRF1) Are Methylated, Respectively, by the Methyltransferases Mtq1p and Mtq2p

“…Although Mtq1p modifies the Mrf1p, the protein that is essential for mitochondrial function (27), the mtq1-⌬ mutant had no significant phenotypes on solid media (Fig. 4).…”

“…The only release factor that acts at the translation termination stop codons in mitochondria is mRF1 (24,26). In the yeast S. cerevisiae, the MRF1 gene encodes a protein more similar to the prokaryotic RF1 than to RF2, and the MRF1 gene is required for proper translation in mitochondria (27). Mrf1p recognizes only the termination codons UAA and UAG on mitochondrial and bacterial ribosomes (25), the same codons recognized by RF1.…”

Mitochondrial ribosomes in cancer

“…However, termination of translation and recycling show stunning deviations from the bacterial pathway [38–41]. For example, mammalian mitochondria utilize only one release factor, mtRF1a [42], to decode all stop codons [43, 44]. …”

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

“…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.…”

“…The MSU1 mutation suppresses only ochre mutations and has occurred at the base of the so called "530-loop" in the 15S rRNA 11. A few years ago, it was shown that the Nam3 protein corresponds to the release factor Mrf1 and the identification of the nam3-1 suppressor mutation was also reported 1.…”