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Peer-reviewedCC BY 4.0The Journal of Biological Chemistry

Cryo-EM structures reveal upstream DNA interactions within the mitochondrial transcription initiation complex

  • Rory E. Sharkey1
  • Caitlin Schroeder2
  • Xiangyu Deng1
  • Jamie Smith1
  • Alfredo J. Hernandez2
  • Yang Gao1
  1. 1Department of BioSciences, Rice University, Houston, Texas, USA
  2. 2Department of Biology, Tufts University, Medford, Massachusetts, USA
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Abstract

Mitochondrial DNA transcription is essential for cellular energy production and is carried out by a streamlined transcription system in which transcription factor A (TFAM), transcription factor B2, and the mitochondrial RNA polymerase (PolRMT) assemble at defined promoters to initiate transcription. Previous structural studies elucidated the core initiation mechanism but relied on truncated promoter templates that excluded upstream regulatory DNA interactions. Here, we present two conformations of mitochondrial transcription initiation complexes assembled on the heavy-strand promote: a TFAM-bound complex with extended upstream DNA and a TFAM-free complex containing short linear DNA. The TFAM-bound structure reveals a transcription-stimulatory interface between PolRMT and the upstream DNA, termed upstream backbone interface, enabled by TFAM-induced promoter bending. Consistent with this structural observation, upstream backbone interface truncation reduces transcription from all mitochondrial DNA promoters, an effect abolished by mutation of the PolRMT interface. In contrast, the TFAM-free structure reveals a transcription-inhibitory interaction of linear upstream DNA with the PolRMT tether helix, which would sterically clash with TFAM binding. Deletion of the tether helix increases off-target transcription, supporting an autoinhibitory role that enhances promoter specificity. Together, these findings reveal how interactions of TFAM and PolRMT with upstream DNA influence activity and specificity of mitochondrial transcription initiation.

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Cite

BibTeXRIS
BibTeX
@article{sharkey2026cryoem,
  title = {{Cryo-EM structures reveal upstream DNA interactions within the mitochondrial transcription initiation complex}},
  author = {Rory E. Sharkey and Caitlin Schroeder and Xiangyu Deng and Jamie Smith and Alfredo J. Hernandez and Yang Gao},
  journal = {The Journal of Biological Chemistry},
  year = {2026},
  month = sep,
  doi = {10.1016/j.jbc.2026.113611},
  url = {https://doi.org/10.1016/j.jbc.2026.113611},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Cryo-EM structures reveal upstream DNA interactions within the mitochondrial transcription initiation complex
AU  - Rory E. Sharkey
AU  - Caitlin Schroeder
AU  - Xiangyu Deng
AU  - Jamie Smith
AU  - Alfredo J. Hernandez
AU  - Yang Gao
PY  - 2026
DA  - 2026/09/29
JO  - The Journal of Biological Chemistry
DO  - 10.1016/j.jbc.2026.113611
UR  - https://doi.org/10.1016/j.jbc.2026.113611
AB  - Mitochondrial DNA transcription is essential for cellular energy production and is carried out by a streamlined transcription system in which transcription factor A (TFAM), transcription factor B2, and the mitochondrial RNA polymerase (PolRMT) assemble at defined promoters to initiate transcription. Previous structural studies elucidated the core initiation mechanism but relied on truncated promoter templates that excluded upstream regulatory DNA interactions. Here, we present two conformations of mitochondrial transcription initiation complexes assembled on the heavy-strand promote: a TFAM-bound complex with extended upstream DNA and a TFAM-free complex containing short linear DNA. The TFAM-bound structure reveals a transcription-stimulatory interface between PolRMT and the upstream DNA, termed upstream backbone interface, enabled by TFAM-induced promoter bending. Consistent with this structural observation, upstream backbone interface truncation reduces transcription from all mitochondrial DNA promoters, an effect abolished by mutation of the PolRMT interface. In contrast, the TFAM-free structure reveals a transcription-inhibitory interaction of linear upstream DNA with the PolRMT tether helix, which would sterically clash with TFAM binding. Deletion of the tether helix increases off-target transcription, supporting an autoinhibitory role that enhances promoter specificity. Together, these findings reveal how interactions of TFAM and PolRMT with upstream DNA influence activity and specificity of mitochondrial transcription initiation.
SN  - 0021-9258
SN  - 1083-351X
ER  -