Abstract
In vitro assay based on a reconstituted mitochondrial transcription system serves as a method of choice to probe the functional importance of proteins and their structural motifs. Here we describe protocols for transcription assays designed to probe activity of the human mitochondrial RNA polymerase and the transcription initiation complex using RNA–DNA scaffold and synthetic promoter templates.
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References
Clayton D (2000) Transcription and replication of mitochondrial DNA. Hum Reprod Embryol 15:11–17
Morozov YI, Parshin AV, Agaronyan K et al (2015) A model for transcription initiation in human mitochondria. Nucleic Acids Res 43:3726–3735
Agaronyan K, Morozov YI, Anikin M, Temiakov D (2015) Replication-transcription switch in human mitochondria. Science 347:548–551
Falkenberg M, Gaspari M, Rantanen A et al (2002) Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA. Nat Genet 31:289–294
Sologub M, Litonin D, Anikin M et al (2009) TFB2 is a transient component of the catalytic site of the human mitochondrial RNA polymerase. Cell 139:934–944
Lodeiro MF, Uchida AU, Arnold JJ et al (2010) Identification of multiple rate-limiting steps during the human mitochondrial transcription cycle in vitro. J Biol Chem 285:16387–16402
Morozov YI, Agaronyan K, Cheung ACM et al (2014) A novel intermediate in transcription initiation by human mitochondrial RNA polymerase. Nucleic Acids Res 42:3884–3893
IDT https://www.idtdna.com/pages/education/decoded/article/annealing-oligonucleotides. Accessed 25 Sep 2018
Shi Y, Dierckx A, Wanrooij PH et al (2012) Mammalian transcription factor A is a core component of the mitochondrial transcription machinery. Proc Natl Acad Sci 109:16510–16515
Acknowledgements
We thank past and present members of the Temiakov laboratory. This work was funded by NIH R35 GM131832.
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Sarfallah, A., Temiakov, D. (2021). In Vitro Reconstitution of Human Mitochondrial Transcription. In: Minczuk, M., Rorbach, J. (eds) Mitochondrial Gene Expression. Methods in Molecular Biology, vol 2192. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0834-0_3
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DOI: https://doi.org/10.1007/978-1-0716-0834-0_3
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-0834-0
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