Abstract
The general features of the organization of in vivo transcription of the mammalian mitochondrial DNA (mtDNA) were delineated in the 1970s and early 1980s by a detailed analysis of the mapping and metabolic properties of the transcripts of the two strands of this DNA (1, 2), the elucidation of the RNA processing events (3), and the identification of the initiation sites for transcription (4, 5). A significant advance in our understanding of the mechanism and regulation of this process was made possible by the development in the past ten years of in vitro transcription systems. Although the “in organello” transcription system (6) has provided an approach for investigating the role of the extramitochondrial environment and the energetic requirements for transcription, the “open” systems have opened the way for the dissection of the enzymology and the detailed mechanistic aspects of this process. Two such systems have been established. One utilizes semipurified protein components to promote transcription from appropriate mtDNA templates (7), whereas the other makes use of a mitochondrial lysate programmed by exogenous templates (8) and supplemented, when necessary, with purified transcription termination factor (mTERF) (9). The rationale behind the latter approach is to reproduce as closely as possible the in vivo situation to support the various steps of the transcription process, under conditions amenable to external manipulation. In this chapter, we describe in detail the protocols used in our laboratory for the study of transcription initiation and termination in a mitochondrial lysate from HeLa cells.
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Ojala, D., Merkel, C., Gelfand, R., and Attardi, G. (1980) The tRNA gene punctuate the reading of genetic information in human mitochondrial DNA. Cell 22, 393–403.
Gelfand, R. and Attardi, G. (1981) Synthesis and turnover of mitochondrial ribonucleic acid in HeLa cells: the mature ribosomal and messenger ribonucleic acid species are metabolically unstable. Mol. Cell. Biol. 1, 497–511.
Ojala, D., Montoya, J., and Attardi, G. (1982) The tRNA punctuation model of RNA processing in human mitochondria. Nature 290, 470–474.
Montoya, J., Christianson, T., Levens, D., Rabinowitz, M., and Attardi, G. (1982) Identification of initiation sites for heavy-strand and light-strand transcription in human mitochondrial DNA. Proc. Natl. Acad. Sci. USA 79, 7195–7199.
Yoza, B. K. and Bogenhagen, D. F. (1984) Identification and in vitro capping of a primary transcript of human mitochondrial DNA. J. Biol. Chem. 259, 3909–3915.
Gaines, G. and Attardi, G. (1984) Highly efficient RNA-synthesizing system that uses isolated human mitochondria: new initiation events and in viva-like processing patterns. Mol. Cell. Biol. 4, 1605–1617.
Walberg, M. W. and Clayton, D. A. (1983) In vitro transcription of human mitochondrial DNA. J. Biol. Chem. 258, 1268–1275.
Shuey, D. J. and Attardi, G. (1985) Characterization of an RNA polymerase activity from HeLa cell nntochondria, which initiates transcription at the heavy strand rRNA promoter in human mitochondrial DNA. J. Biol. Chem. 260, 1952–1958.
Kruse, B., Narasimhan, N., and Attardi, G. (1989) Termination of transcription in human mitochondria: identification and purification of a DNA binding protein factor that promotes termination. Cell 58, 391–397.
Puck, T. T., and Fisher, H. W. (1956) Genetics of somatic mammalian cells. I. Demonstration of the existence of mutants with different growth requirements in a human cancer cell strain (HeLa) J. Exp. Med. 104, 427–434
Levintov, L. and Darnell, J. E. (1960) A simplified procedure for purification of large amounts of poliovirus: characterization and amino acid analysis of type 1 poliovirus. J. Biol. Chem. 235, 70–73.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Bradford, M. M. (1976) A rapid and sensitive method of the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248–254.
Narasimhan, N. and Attardi, G. (1987) Specific requirements for ATP at an early step of in vitro transcription of human mitochondrial DNA. Proc. Natl. Acad. Sci. USA 84, 4078–4082.
Martin, S. A., Paoletti, E., and Moss, B. (1975) Purification of the mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions. J. Biol. Chem. 250, 9322–9329.
Gaines, G. and King, M. P. unpublished data.
Anderson, S., Bankier, A. T., Barrell, B. G., deBruijn, M. H. L., Coulson, A. R., Drouin, J., Eperon, I. E., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Stadler, R., and Young, I. G. (1981) Sequence and organization of the human mitochondrial genome. Nature (Lond.) 290, 457–465.
King, M. P. (1987) PhD. Thesis, California Institute of Technology, Pasadena, CA.
Attardi, G., Cantatore, P., Chomyn, A., Crews, S., Gelfand, R., Merkel, C., Montoya, J., and Ojala, D. (1982) A comprehensive view of mitochondrial gene expression in human cells, in Mitochondrial Genes (Slonimski, P., Borst, P., and Attardi, G., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 51–71.
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© 1995 Humana Press Inc.
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Kruse, B., Murdter, N.N., Attardi, G. (1995). Transcription System Using a HeLa Cell Mitochondrial Lysate. In: Tymms, M.J. (eds) In Vitro Transcription and Translation Protocols. Methods in Molecular Biology, vol 37. Humana Press. https://doi.org/10.1385/0-89603-288-4:179
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DOI: https://doi.org/10.1385/0-89603-288-4:179
Publisher Name: Humana Press
Print ISBN: 978-0-89603-288-0
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