TFAM's Contributions to mtDNA Replication and OXPHOS Biogenesis Are Genetically Separable

Area of Science:

• Mitochondrial biology
• Molecular genetics
• Biochemistry

Background:

• Human mitochondrial transcription factor A (hTFAM) is essential for mitochondrial DNA (mtDNA) replication and cellular respiration.
• Animal TFAM orthologs exhibit varied abilities to support hmtDNA replication, not correlating with phylogenetic proximity or sequence similarity.
• Chicken TFAM (chTFAM) fails to support hmtDNA replication, contrasting with TFAM from the coelacanth.

Purpose of the Study:

• To investigate the functional divergence of TFAM orthologs using reverse genetics.
• To elucidate the specific roles of TFAM in mtDNA replication and respiratory chain biogenesis.
• To identify key TFAM residues critical for its functions.

Main Methods:

• GeneSwap approach for reverse genetic analysis of chTFAM.
• Creation of "humanized" chTFAM variants by modifying DNA-contacting or variable residues.
• Assessment of mtDNA copy number (mtCN) and cellular respiration in cells expressing engineered TFAM variants.

Main Results:

• Two chTFAM variants, Ch13 and Ch22, were generated with distinct effects on mtCN and respiration.
• Ch13 exhibited low mtCN but robust respiration, while Ch22 showed the opposite phenotype.
• These variants demonstrated complementary effects, and similar opposing trends were observed with frog TFAM.
• Evidence suggests TFAM residues involved in DNA contact are primary drivers of mtDNA replication.

Conclusions:

• TFAM's roles in mtDNA replication and respiratory chain biogenesis are genetically separable.
• Specific TFAM residues interacting with DNA are critical for efficient mtDNA replication.
• A novel regulatory mechanism for respiratory chain biogenesis involving rRNA supply was identified.