Abstract
Characterization of the basic transcription machinery of mammalian mitochondrial DNA has been greatly supported by the availability of pure recombinant mitochondrial RNA polymerase (mtRNAP) and accessory factors, which allowed to develop a reconstituted in vitro transcription system. This chapter outlines a general strategy that makes use of a minimal promoter-independent transcription assay to study mitochondrial transcription termination in animal systems. We used such a system to investigate the transcription termination properties of the sea urchin factor mtDBP, however, it is applicable to the study of transcription termination in a variety of organisms, provided that the pure mtRNAP and the transcription termination factor are available.
The assay here described contains the recombinant proteins mtRNAP and mtDBP, both expressed in insect cells, and a template consisting of a 3′-tailed DNA construct bearing the sequence bound by mtDBP. Transcription by the RNA polymerase produces run-off and terminated molecules, the size of the latter being consistent with RNA chain arrest in correspondence of the mtDBP–DNA complex. Transcription termination is protein-dependent as addition of increasing amounts of mtDBP to the assay causes a decrease in the intensity of the run-off and the gradual appearance of short-terminated molecules. Furthermore, we report a method, based on pulse-chase experiments, which allows us to distinguish between the true termination and the pausing events.
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References
Montoya J, Lopez Perez MJ, Ruiz-Pesini E (2006) Mitochondrial DNA transcription and disease: past present and future. Biochim. Biophys. Acta 1757, 11179–1189.
Falkenberg M, Larsson NG, Gustafsson CM (2007) DNA replication and transcription in mammalian mitochondria. Annu. Rev. Biochem. 76, 679–699.
Dubrovsky EB, Dubrovskaya VA, Levinger L, Schiffer S, Marchfelder A (2004) Drosophila RNase Z processes mitochondrial and nuclear pre-tRNA 3′ ends in vivo. Nucleic Acids Res. 32, 255–262.
Cantatore P, Roberti M, Loguercio Polosa P, Mustich A, Gadaleta MN (1990) Mapping and characterization of Paracentrotus lividus mitochondrial transcripts: multiple and overlapping transcription units. Curr. Genet. 17, 235–245.
Berthier F, Reanaud M, Alziari S, Durand R (1986) RNA mapping on Drosophila mitochondrial DNA: precursors and template strands. Nucleic Acids Res. 14, 4519–4533
Roberti M, Loguercio Polosa P, Bruni F, Musicco C, Gadaleta MN, Cantatore P (2003) DmTTF, a novel mitochondrial transcription termination factor that recognizes two sequences of Drosophila melanogastermitochondrial DNA, Nucleic Acids Res. 31, 1597–1604.
Carrodeguas JA, Vallejo CG (1997) Mitochondrial transcription initiation in the crustacean Artemia franciscana. Eur. J. Biochem. 250, 514–523.
Park CB, Asin-Cayuela J, Camara Y, Shi Y, Pellegrini M, Gaspari M, Wibom R, Hultenby K, Erdjument-Bromage H, Tempst P, Falkenberg M, Gustafsson CM, Larsson NG (2007) MTERF3 is a negative regulator of mammalian mtDNA transcription. Cell. 130, 273–285.
Kruse B, Narashiman N, Attardi G (1989) Termination of transcription in human mitochondria: identification and purification of a DNA binding protein that promotes termination. Cell. 58, 391–397.
Martin M, Cho J, Cesare AJ, Griffith JD, Attardi G (2005) Termination factor-mediated DNA loop between termination and initiation sites drives mitochondrial rRNA synthesis. Cell. 123, 1227–1240
Loguercio Polosa P, Roberti M, Musicco C, Gadaleta, MN, Quagliariello E, Cantatore P (1999) Cloning and characterisation of mtDBP, a DNA-binding protein which binds two distinct regions of sea urchin mitochondrial DNA. Nucleic Acids Res., 27, 1890–1899.
Loguercio Polosa P, Deceglie S, Roberti M, Gadaleta MN, Cantatore P (2005) Contrahelicase activity of the mitochondrial transcription termination factor mtDBP. Nucleic Acids Res. 33, 3812–3820.
Roberti M, Bruni F, Loguercio Polosa P, Gadaleta MN, Cantatore P (2006) The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription. Nucleic Acids Res. 34, 2109–2116.
Roberti M, Fernandez-Silva P, Loguercio Polosa P, Fernandez Vizarra E, Bruni F, Deceglie S, Montoya J, Gadaleta MN, Cantatore P (2005) In vitro transcription termination activity of the Drosophila mitochondrial DNA-binding protein DmTTF, Biochem. Biophys. Res. Commun. 331, 357–362.
Roberti M, Bruni F, Loguercio Polosa P, Manzari C, Gadaleta MN, Cantatore P (2006). The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription. Nucleic Acids Res. 34, 2109–2116.
Loguercio Polosa P, Deceglie S, Falkenberg M, Roberti M, Di Ponzio B, Gadaleta MN, Cantatore P (2007) Cloning of the sea urchin mitochondrial RNA polymerase and reconstitution of the transcription termination system. Nucleic Acids Res. 35, 2413–2427.
Falkenberg M, Gaspari M, Rantanen A, Trifunovic A, Larsson NG, Gustafsson CM (2002) Mitochondrial transcription factors B1 and B2 activate transcription of human mtDNA. Nat. Genet. 31, 289–294.
Sambrook J, Russel DW (2001) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor New York.
Dedrick RL, Chamberlin MJ (1985) Studies on transcription of 3’-extended templates by mammalian RNA polymerase II. Parameters that affect the initiation and elongation reactions. Biochemistry, 24, 2245–2253.
Micol V, Fernandez-Silva P, Attardi G (1996) Methods Enzymol. Academic Press 264, 158–173.
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Polosa, P.L., Deceglie, S., Roberti, M., Gadaleta, M.N., Cantatore, P. (2009). Methods for Studying Mitochondrial Transcription Termination with Isolated Components. In: Stuart, J.A. (eds) Mitochondrial DNA. Methods in Molecular Biology™, vol 554. Humana Press. https://doi.org/10.1007/978-1-59745-521-3_9
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DOI: https://doi.org/10.1007/978-1-59745-521-3_9
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