Department of Physiology and Cell Biology, University of South Alabama, Mobile, AL 36688, USA.
Cells. 2022 Nov 24;11(23):3754. doi: 10.3390/cells11233754.
The ability of animal orthologs of human mitochondrial transcription factor A (hTFAM) to support the replication of human mitochondrial DNA (hmtDNA) does not follow a simple pattern of phylogenetic closeness or sequence similarity. In particular, TFAM from chickens (, chTFAM), unlike TFAM from the "living fossil" fish coelacanth (), cannot support hmtDNA replication. Here, we implemented the recently developed GeneSwap approach for reverse genetic analysis of chTFAM to obtain insights into this apparent contradiction. By implementing limited "humanization" of chTFAM focused either on amino acid residues that make DNA contacts, or the ones with significant variances in side chains, we isolated two variants, Ch13 and Ch22. The former has a low mtDNA copy number (mtCN) but robust respiration. The converse is true of Ch22. Ch13 and Ch22 complement each other's deficiencies. Opposite directionalities of changes in mtCN and respiration were also observed in cells expressing frog TFAM. This led us to conclude that TFAM's contributions to mtDNA replication and respiratory chain biogenesis are genetically separable. We also present evidence that TFAM residues that make DNA contacts play the leading role in mtDNA replication. Finally, we present evidence for a novel mode of regulation of the respiratory chain biogenesis by regulating the supply of rRNA subunits.
动物与人线粒体转录因子 A（hTFAM）的同源物支持人线粒体 DNA（hmtDNA）复制的能力并不遵循简单的系统发育或序列相似性模式。特别是，来自鸡（chTFAM）的 TFAM 与来自“活化石”鱼类腔棘鱼（）的 TFAM 不同，不能支持 hmtDNA 复制。在这里，我们实施了最近开发的 GeneSwap 方法，用于 chTFAM 的反向遗传分析，以深入了解这种明显的矛盾。通过实施针对 DNA 接触的氨基酸残基或侧链有显著差异的氨基酸残基的有限“人源化”，我们分离出两个变体，Ch13 和 Ch22。前者 mtDNA 拷贝数（mtCN）低，但呼吸作用稳健。Ch22 则相反。Ch13 和 Ch22 相互补充彼此的缺陷。在表达蛙 TFAM 的细胞中也观察到 mtCN 和呼吸作用变化的相反方向性。这使我们得出结论，TFAM 对 mtDNA 复制和呼吸链生物发生的贡献在遗传上是可分离的。我们还提供了证据表明，与 DNA 接触的 TFAM 残基在 mtDNA 复制中起主导作用。最后，我们提出了一种通过调节 rRNA 亚基供应来调节呼吸链生物发生的新型调节模式的证据。
