Co-evolution of bizarre armless tRNAs and their maturation enzymes in Enoplea

在后生动物的线粒体中，许多tRNA显示出与标准结构的偏差，导致不寻常和奇异的转录本。在Enoplea线粒体中，我们最近发现了显著减少的无臂tRNA，代表了这种RNA类型的最极端形式。为了识别这些转录物作为底物，线粒体tRNA成熟和氨酰化活性必须经历特定的适应。使用Romanomermis culicivorax作为模式生物，我们将表征相应的线粒体CCA添加酶，RNase P以及几个氨酰-tRNA合成酶作为重组蛋白。虽然后者的活动代表了专门的线粒体酶版本，但CCA添加酶的单一形式负责细胞溶质（典型）和线粒体（奇异）tRNA的成熟。因此，这种酶必须与其底物进行不寻常的共同进化。利用动力学表征、竞争研究和酶嵌合体的分析，我们希望在酶的功能域中识别这种适应。此外，单独的R.将比较用于tRNA前体转录物加工和氨酰化的食culicivorax酶的底物特异性与作用于常规tRNA的活性。最后，结构分析，如SHAPE，在线探测以及无臂tRNA的NMR研究将确定这些奇怪的转录本和它们的成熟酶之间相互作用所需的特定特征。这些分析将详细了解tRNA加工酶与其底物相互作用的灵活性和适应性。

In mitochondria of metazoans, many tRNAs show deviations from the standard structure, resulting in unusual and bizarre transcripts. In Enoplea mitochondria, we could recently identify dramatically reduced armless tRNAs, representing the most extreme form of this RNA type. To recognize these transcripts as substrates, mitochondrial tRNA maturation and aminoacylation activities must have undergone a specific adaptation. Using Romanomermis culicivorax as a model organism, we will characterize the corresponding mitochondrial CCA-adding enzyme, RNase P as well as several aminoacyl-tRNA synthetases as recombinant proteins. While the latter activities represent specialized mitochondrial enzyme versions, a single form of the CCA-adding enzyme is responsible for the maturation of both cytosolic (canonical) and mitochondrial (bizarre) tRNAs. Hence, this enzyme had to undergo an unusual co-evolution with its substrates. Using kinetic characterization, competition studies and the analysis of enzyme chimeras, we want identify this adaptation in the functional domains of the enzyme. In addition, individual R. culicivorax enzymes for tRNA precursor transcript processing and aminoacylation will be compared in their substrate specificity to activities acting on conventional tRNAs. Finally, structure analyses like SHAPE, in line probing as well as NMR studies of armless tRNAs will identify specific features required for the interplay between these bizarre transcripts and their maturation enzymes. These analyses will give detailed insights into the flexibility and adaptability of tRNA processing enzymes concerning the interaction with their substrates.