Translational activators align mitochondrial mRNAs at the small ribosomal subunit for translation initiation
Mitochondrial gene expression is essential for oxidative phosphorylation. Mitochondrial-encoded mRNAs are translated by dedicated mitochondrial ribosomes (mitoribosomes), whose regulation remains elusive. In the baker´s yeast Saccharomyces cerevisiae, nuclear-encoded mitochondrial translational activators (TAs) facilitate transcript-specific translation by a yet unknown mechanism. Here, we investigated the function of TAs containing RNA-binding pentatricopeptide repeats (PPRs) using selective mitoribosome profiling and cryo-EM structural analysis. These analyses revealed that TAs exhibit strong selectivity for mitoribosomes initiating on their target transcripts. Moreover, TA-mitoribosome footprints indicated that TAs recruit mitoribosomes proximal to the start codon. Two cryo-EM structures of mRNA-TA complexes bound to post-initiation/pre-elongation-stalled mitoribosomes revealed the general mechanism of TA action. Specifically, the TAs bind to structural elements in the 5' UTR of the client mRNA as well as to the mRNA exit channel to align the mRNA in the small subunit during initiation. Our findings provide a structural basis for understanding how mitochondria achieve transcript-specific translation initiation without relying on general sequence elements to position ribosomes at start codons. Overall design: Mitochondrial selective ribosome profiling of translational activators and related factors in S. cerevisiae cells