The C-Terminal Tail of Mitochondrial Transcription Factor A Is Dispensable for Mitochondrial DNA Replication and Transcription In Situ

Abstract

Mitochondrial transcription factor A (TFAM) is one of the widely studied but still incompletely understood mitochondrial protein, which plays a crucial role in the maintenance and transcription of mitochondrial DNA (mtDNA). The available experimental evidence is often contradictory in assigning the same function to various TFAM domains, partly owing to the limitations of those experimental systems. Recently, we developed the GeneSwap approach, which enables in situ reverse genetic analysis of mtDNA replication and transcription and is devoid of many of the limitations of the previously used techniques. Here, we utilized this approach to analyze the contributions of the TFAM C-terminal (tail) domain to mtDNA transcription and replication. We determined, at a single amino acid (aa) resolution, the TFAM tail requirements for in situ mtDNA replication in murine cells and established that tail-less TFAM supports both mtDNA replication and transcription. Unexpectedly, in cells expressing either C-terminally truncated murine TFAM or DNA-bending human TFAM mutant L6, HSP1 transcription was impaired to a greater extent than LSP transcription. Our findings are incompatible with the prevailing model of mtDNA transcription and thus suggest the need for further refinement.

35 Years of TFAM Research: Old Protein, New Puzzles

“…Recently, it has been demonstrated that the TFAM tail is dispensable for mtDNA replication and transcription in situ. This evidence represents a challenge to the current model of mtDNA transcription, which postulates a critical role for the TFAM tail in the assembly of the mitochondrial transcription apparatus [ 43 ].…”

“…However, recent evidence from the higher-order structures of the transcriptional apparatus indicates that TFAM binds in the same orientation at the LSP and HSP1 [ 42 ]. Therefore, it is unclear why TFAM bending mutants have a greater effect on LSP transcription than on HSP1 transcription in vitro [ 36 , 50 ], or why the effects of bending mutations on mitochondrial promoters are reversed in situ [ 43 ].…”

“…Previously, it was suggested that TFAM might mediate the assembly of transcription complexes through interactions of its C-terminal tail with TFB2M [ 61 ]. Recent in situ data indicate that the TFAM tail is dispensable for mitochondrial transcription, suggesting that it is unlikely that the TFAM tail plays a critical role in the assembly of the mitochondrial transcription apparatus [ 43 ]. Therefore, it appears to be more likely that TFAM HMG2–POLRMT interactions drive the assembly of transcription complexes [ 42 ].…”

“…Note that, despite the evidence supporting the existence of HSP2 coming from the identification of transcripts with distinct 5’ ends corresponding to this promoter [ 65 , 114 , 115 ], reconstitution of HSP2 transcription in vitro [ 105 , 106 ], and proof from recent RNAseq studies [ 116 ], the existence of HSP2 is not universally accepted, primarily due to this promoter’s weakness and unusual location [ 117 ]. However, TFAM C-terminal truncations and mutations differentially affect the steady-state levels of HSP1 and HSP2 transcripts [ 25 , 43 , 118 ]. Collectively, these observations lend further credence to the existence of two HSPs and mechanistically distinct contributions of TFAM to the activities of these two promoters.…”

TFAM in mtDNA Homeostasis: Open Questions

“…In vitro, LSP transcription appears to depend on TFAM, particularly on its C-terminus [ 7 ]. However, more recent evidence has suggested that tail-less TFAM can initiate LSP transcription on longer DNA templates [ 8 , 9 ] and can support mtDNA transcription and replication in situ [ 10 ]. Notably, the initiation of the lagging mtDNA strand replication appears to be TFAM-independent [ 11 , 12 ].…”