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Abstract

The genes in mitochondrial DNA code for essential subunits of the respiratory chain complexes. In yeast, expression of mitochondrial genes is controlled by a group of gene-specific translational activators encoded in the nucleus. These factors appear to be part of a regulatory system that enables concerted expression of the necessary genes from both nuclear and mitochondrial genomes to produce functional respiratory complexes. Many of the translational activators are believed to act on the 5'-untranslated regions of target mRNAs, but the molecular mechanisms involved in this regulation remain obscure. In this study, we used a combination of in vivo and in vitro analyses to characterize the interactions of one of these translational activators, the pentatricopeptide repeat (PPR) protein Pet111p, with its presumed target, COX2 mRNA, which encodes subunit II of cytochrome c oxidase. Using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis, we found that Pet111p binds directly and specifically to a 5'-end proximal region of the COX2 transcript. Further, we applied in vitro RNase footprinting and mapped two binding targets of the protein, one of which is located in the 5'-untranslated leader and the other within the coding sequence. Combined with available genetic data, these results suggest a plausible mechanism of translational activation, in which binding of Pet111p may prevent inhibitory secondary structures from forming in the translation initiation region, thus rendering the mRNA available for interaction with the ribosome.