Mammalian mitochondrial DNA (mtDNA) is packaged by mitochondrial transcription factor A (TFAM) into mitochondrial nucleoids that are of key importance in controlling the transmission and expression of mtDNA. Nucleoid ultrastructure is poorly defined, and therefore we used a combination of biochemistry, superresolution microscopy, and electron microscopy to show that mitochondrial nucleoids have an irregular ellipsoidal shape and typically contain a single copy of mtDNA. Rotary shadowing electron microscopy revealed that nucleoid formation in vitro is a multistep process initiated by TFAM aggregation and cross-strand binding. Superresolution microscopy of cultivated cells showed that increased mtDNA copy number increases nucleoid numbers without altering their sizes. Electron cryo-tomography visualized nucleoids at high resolution in isolated mammalian mitochondria and confirmed the sizes observed by superresolution microscopy of cell lines. We conclude that the fundamental organizational unit of the mitochondrial nucleoid is a single copy of mtDNA compacted by TFAM, and we suggest a packaging mechanism.nucleoids | mitochondria | cryo-ET | STED | nanoscopy

Mitochondrial transcription for replication primer formation has priority over gene expression at low POLRMT levels.

A single-subunit RNA polymerase, POLRMT, transcribes the mitochondrial genome in human cells. Recently, a factor termed as the mitochondrial transcription elongation factor, TEFM, was shown to stimulate transcription elongation in vivo, but its effect in vitro was relatively modest. In the current work, we have isolated active TEFM in recombinant form and used a reconstituted in vitro transcription system to characterize its activities. We show that TEFM strongly promotes POLRMT processivity as it dramatically stimulates the formation of longer transcripts. TEFM also abolishes premature transcription termination at conserved sequence block II, an event that has been linked to primer formation during initiation of mtDNA synthesis. We show that POLRMT pauses at a wide range of sites in a given DNA sequence. In the absence of TEFM, this leads to termination; however, the presence of TEFM abolishes this effect and aids POLRMT in continuation of transcription. Further, we show that TEFM substantially increases the POLRMT affinity to an elongation-like DNA:RNA template. In combination with previously published in vivo observations, our data establish TEFM as an essential component of the mitochondrial transcription machinery.

Regulation of replication and expression of mitochondrial DNA (mt DNA ) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor ( TEFM ) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome‐length transcription for mt DNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter‐distal mitochondrial transcripts are drastically reduced in conditional Tefm ‐knockout hearts. In contrast, the promoter‐proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mt DNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity‐labeling (Bio ID ) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.

Mammalian mitochondrial transcription is executed by a single subunit mitochondrial RNA polymerase (Polrmt) and its two accessory factors, mitochondrial transcription factors A and B2 (Tfam and Tfb2m). Polrmt is structurally related to single-subunit phage RNA polymerases, but it also contains a unique N-terminal extension (NTE) of unknown function. We here demonstrate that the NTE functions together with Tfam to ensure promoter-specific transcription. When the NTE is deleted, Polrmt can initiate transcription in the absence of Tfam, both from promoters and non-specific DNA sequences. Additionally, when in presence of Tfam and a mitochondrial promoter, the NTE-deleted mutant has an even higher transcription activity than wild-type polymerase, indicating that the NTE functions as an inhibitory domain. Our studies lead to a model according to which Tfam specifically recruits wild-type Polrmt to promoter sequences, relieving the inhibitory effect of the NTE, as a first step in transcription initiation. In the second step, Tfb2m is recruited into the complex and transcription is initiated.