Transcription from the second heavy-strand promoter of human mtDNA is repressed by transcription factor A in vitro

Abstract

Cell-based studies support the existence of two promoters on the heavy strand of mtDNA: heavy-strand promoter 1 (HSP1) and HSP2. However, transcription from HSP2 has been reported only once in a cell-free system, and never when recombinant proteins have been used. Here, we document transcription from HSP2 using an in vitro system of defined composition. An oligonucleotide template representing positions 596-685 of mtDNA was sufficient to observe transcription by the human mtRNA polymerase (POLRMT) that was absolutely dependent on mitochondrial transcription factor B2 (TFB2M). POLRMT/TFB2M-dependent transcription was inhibited by concentrations of mitochondrial transcription factor A (TFAM) stoichiometric with the transcription template, a condition that activates transcription from the light-strand promoter (LSP) in vitro. Domains of TFAM required for LSP activation were also required for HSP2 repression, whereas other mtDNA binding proteins failed to alter transcriptional output. Binding sites for TFAM were located on both sides of the start site of transcription from HSP2, suggesting that TFAM binding interferes with POLRMT and/or TFB2M binding. Consistent with a competitive binding model for TFAM repression of HSP2, the impact of TFAM concentration on HSP2 transcription was diminished by elevating the POLRMT and TFB2M concentrations. In the context of our previous studies of LSP and HSP1, it is now clear that three promoters exist in human mtDNA. Each promoter has a unique requirement for and/or response to the level of TFAM present, thus implying far greater complexity in the regulation of mammalian mitochondrial transcription than recognized to date. mitochondria | gene expression | initiation | gel-shift assay | footprinting

Maintenance and Expression of Mammalian Mitochondrial DNA

“…There is no consensus on whether the HSP2 promoter exists. Some reports have failed to observe initiation from HSP2 in reconstituted transcription systems, whereas others have suggested that transcription initiation at HSP2 can be observed in vitro (30,(56)(57)(58)(59). However, the data are not congruent because the precise HSP2 transcription start site mapped in vitro differs from the one that has been mapped in vivo (55,59).…”

Structural models of mammalian mitochondrial transcription factor B2

“…inhibited by TFAM [12,13]. These recent observations suggest far more regulation of mitochondrial transcription than previously imagined.…”

Deceleration of liver regeneration by knockdown of augmenter of liver regeneration gene is associated with impairment of mitochondrial DNA synthesis in mice

“…PGC-1␣ is a key transcriptional regulator of cellular energy metabolism to stimulate mitochondrial biogenesis (3). TFAM is capable of binding and wrapping mtDNA, as well as activating and regulating mtDNA transcription and replication (8,30,37). TFAM plays a key role in mitochondrial biogenesis, since it is essential to the initiation of both the replication and expression of mtDNA, which encodes portions of the mitochondrial proteins, such as cytochrome c oxidase II (13).…”