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J. Biol. Chem., Vol. 279, Issue 14, 14096-14103, April 2, 2004

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

Joanna Towpik, Agnieszka Chaciñska, Malgorzata Ciela, Krzysztof Ginalski¶, and Magdalena Boguta||

From the Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawiñskiego 5A, 02-106 Warsaw, Poland, Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, and ^¶BioInfoBank Institute, Limanowskiego 24A, 60-744 Poznañ, Poland

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.

Received for publication, November 25, 2003 , and in revised form, January 16, 2004.

^* This work was supported by Grant 3PO4A03922 from the Committee of Scientific Research (to K. B. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 4822-592-1312; Fax: 4822-658-4636; E-mail: magda{at}poczta.ibb.waw.pl.

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