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.线粒体基因表达对于氧化磷酸化至关重要。线粒体编码的mRNA由专属的线粒体核糖体（mitoribosomes）进行翻译，而其调控机制仍未阐明。在酿酒酵母（Saccharomyces cerevisiae）中，核编码的线粒体翻译激活因子（TAs）以尚不明确的机制促进转录本特异性翻译。本研究借助选择性线粒体核糖体谱分析与冷冻电镜（cryo-EM）结构解析，探究了含有RNA结合型五肽重复序列（PPRs）的TAs的功能。分析结果显示，TAs对在其靶转录本上起始翻译的线粒体核糖体具有极强的选择性。此外，TA-线粒体核糖体足迹分析表明，TAs可将线粒体核糖体招募至起始密码子附近。我们解析了结合有mRNA-TA复合物的起始后/延伸前停滞线粒体核糖体的两种冷冻电镜结构，揭示了TAs发挥作用的通用机制。具体而言，TAs可结合靶标mRNA 5'非翻译区（5' UTR）的结构元件以及mRNA出口通道，从而在翻译起始过程中将mRNA在核糖体小亚基上精准排布。本研究的发现为理解线粒体如何在不依赖通用序列元件将核糖体定位至起始密码子的情况下实现转录本特异性翻译起始提供了结构基础。