SINGLE-POINT MUTATIONS IN DOMAIN-II OF THE YEAST MITOCHONDRIAL RELEASE FACTOR MRF-1 AFFECT RIBOSOME BINDING

We have recently described two yeast strains that are mutated in the M RF1 gene encoding the mitochondrial release factor mRF-1. Both mutants provoke gene-specific defects in mitochondrial translational terminat ion. In the present study we report the cloning, sequencing, as well a s an analysis of residual activities of both mutant mrf1 alleles. Each allele specifies a different single amino acid substitution located o ne amino acid apart. The amino acid changes do not affect the level or cellular localization of the mutant proteins, since equal amounts of wild type and mutant mRF-1 were detected in the mitochondrial compartm ent. Over-expression of the mutant alleles in wild type and mrf1 mutan t yeast strains produces a phenotype consistent with a reduced affinit y of the mutant release factors for the ribosome, indicating that the mutations map in a release factor domain involved in ribosome binding. We also demonstrate that nonsense suppression caused by a mutation in the mitochondrial homolog of the E.coli small ribosomal protein S4 ca n be reversed by a slight over-expression of the MRF1 gene.