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. 1991 Apr;11(4):2035–2039. doi: 10.1128/mcb.11.4.2035

Accurate transcription of a plant mitochondrial gene in vitro.

P J Hanic-Joyce 1, M W Gray 1
PMCID: PMC359890  PMID: 1848669

Abstract

To investigate transcriptional mechanisms in plant mitochondria, we have developed an accurate and efficient in vitro transcription system consisting of a partially purified wheat mitochondrial extract programmed with cloned DNA templates containing the promoter for the wheat mitochondrial cytochrome oxidase subunit II gene (coxII). Using this system, we localize the coxII promoter to a 372-bp region spanning positions -56 to -427 relative to the coxII translation initiation codon. We show that in vitro transcription of coxII is initiated at position -170, precisely the same site at which transcription is initiated in vivo. Transcription begins within the sequence GTATAGTAAGTA (the initiating nucleotide is underlined), which is similar to the consensus yeast mitochondrial promoter motif, (A/T)TATAAGTA. This is the first in vitro system that faithfully reproduces in vivo transcription of a plant mitochondrial gene.

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Selected References

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  1. Biswas T. K., Getz G. S. Nucleotides flanking the promoter sequence influence the transcription of the yeast mitochondrial gene coding for ATPase subunit 9. Proc Natl Acad Sci U S A. 1986 Jan;83(2):270–274. doi: 10.1073/pnas.83.2.270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Biswas T. K., Getz G. S. Promoter-promoter interactions influencing transcription of the yeast mitochondrial gene, Oli 1, coding for ATPase subunit 9. Cis and trans effects. J Biol Chem. 1988 Apr 5;263(10):4844–4851. [PubMed] [Google Scholar]
  3. Biswas T. K., Ticho B., Getz G. S. In vitro characterization of the yeast mitochondrial promoter using single-base substitution mutants. J Biol Chem. 1987 Oct 5;262(28):13690–13696. [PubMed] [Google Scholar]
  4. Bogenhagen D. F., Applegate E. F., Yoza B. K. Identification of a promoter for transcription of the heavy strand of human mtDNA: in vitro transcription and deletion mutagenesis. Cell. 1984 Apr;36(4):1105–1113. doi: 10.1016/0092-8674(84)90061-8. [DOI] [PubMed] [Google Scholar]
  5. Bogenhagen D. F., Yoza B. K. Accurate in vitro transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters. Mol Cell Biol. 1986 Jul;6(7):2543–2550. doi: 10.1128/mcb.6.7.2543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bonen L., Boer P. H., Gray M. W. The wheat cytochrome oxidase subunit II gene has an intron insert and three radical amino acid changes relative to maize. EMBO J. 1984 Nov;3(11):2531–2536. doi: 10.1002/j.1460-2075.1984.tb02168.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chang D. D., Clayton D. A. Precise assignment of the heavy-strand promoter of mouse mitochondrial DNA: cognate start sites are not required for transcriptional initiation. Mol Cell Biol. 1986 Sep;6(9):3262–3267. doi: 10.1128/mcb.6.9.3262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chang D. D., Clayton D. A. Precise assignment of the light-strand promoter of mouse mitochondrial DNA: a functional promoter consists of multiple upstream domains. Mol Cell Biol. 1986 Sep;6(9):3253–3261. doi: 10.1128/mcb.6.9.3253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chang D. D., Clayton D. A. Precise identification of individual promoters for transcription of each strand of human mitochondrial DNA. Cell. 1984 Mar;36(3):635–643. doi: 10.1016/0092-8674(84)90343-x. [DOI] [PubMed] [Google Scholar]
  10. Chang D. D., Fisher R. P., Clayton D. A. Roles for a promoter and RNA processing in the synthesis of mitochondrial displacement-loop strands. Biochim Biophys Acta. 1987 Jul 14;909(2):85–91. doi: 10.1016/0167-4781(87)90029-7. [DOI] [PubMed] [Google Scholar]
  11. Chang D. D., Hixson J. E., Clayton D. A. Minor transcription initiation events indicate that both human mitochondrial promoters function bidirectionally. Mol Cell Biol. 1986 Jan;6(1):294–301. doi: 10.1128/mcb.6.1.294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Covello P. S., Gray M. W. RNA editing in plant mitochondria. Nature. 1989 Oct 19;341(6243):662–666. doi: 10.1038/341662a0. [DOI] [PubMed] [Google Scholar]
  13. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edwards J. C., Levens D., Rabinowitz M. Analysis of transcriptional initiation of yeast mitochondrial DNA in a homologous in vitro transcription system. Cell. 1982 Dec;31(2 Pt 1):337–346. doi: 10.1016/0092-8674(82)90127-1. [DOI] [PubMed] [Google Scholar]
  15. Fisher R. P., Clayton D. A. A transcription factor required for promoter recognition by human mitochondrial RNA polymerase. Accurate initiation at the heavy- and light-strand promoters dissected and reconstituted in vitro. J Biol Chem. 1985 Sep 15;260(20):11330–11338. [PubMed] [Google Scholar]
  16. Fisher R. P., Parisi M. A., Clayton D. A. Flexible recognition of rapidly evolving promoter sequences by mitochondrial transcription factor 1. Genes Dev. 1989 Dec;3(12B):2202–2217. doi: 10.1101/gad.3.12b.2202. [DOI] [PubMed] [Google Scholar]
  17. Fisher R. P., Topper J. N., Clayton D. A. Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements. Cell. 1987 Jul 17;50(2):247–258. doi: 10.1016/0092-8674(87)90220-0. [DOI] [PubMed] [Google Scholar]
  18. Gray M. W. Origin and evolution of mitochondrial DNA. Annu Rev Cell Biol. 1989;5:25–50. doi: 10.1146/annurev.cb.05.110189.000325. [DOI] [PubMed] [Google Scholar]
  19. Hanic-Joyce P. J., Gray M. W. Processing of transfer RNA precursors in a wheat mitochondrial extract. J Biol Chem. 1990 Aug 15;265(23):13782–13791. [PubMed] [Google Scholar]
  20. Hixson J. E., Clayton D. A. Initiation of transcription from each of the two human mitochondrial promoters requires unique nucleotides at the transcriptional start sites. Proc Natl Acad Sci U S A. 1985 May;82(9):2660–2664. doi: 10.1073/pnas.82.9.2660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kennell J. C., Lambowitz A. M. Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites. Mol Cell Biol. 1989 Sep;9(9):3603–3613. doi: 10.1128/mcb.9.9.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. L'Abbé D., Lang B. F., Desjardins P., Morais R. Histidine tRNA from chicken mitochondria has an uncoded 5'-terminal guanylate residue. J Biol Chem. 1990 Feb 15;265(5):2988–2992. [PubMed] [Google Scholar]
  23. Mulligan R. M., Lau G. T., Walbot V. Numerous transcription initiation sites exist for the maize mitochondrial genes for subunit 9 of the ATP synthase and subunit 3 of cytochrome oxidase. Proc Natl Acad Sci U S A. 1988 Nov;85(21):7998–8002. doi: 10.1073/pnas.85.21.7998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mulligan R. M., Maloney A. P., Walbot V. RNA processing and multiple transcription initiation sites result in transcript size heterogeneity in maize mitochondria. Mol Gen Genet. 1988 Mar;211(3):373–380. doi: 10.1007/BF00425688. [DOI] [PubMed] [Google Scholar]
  25. Osinga K. A., De Vries E., Van der Horst G. T., Tabak H. F. Initiation of transcription in yeast mitochondria: analysis of origins of replication and of genes coding for a messenger RNA and a transfer RNA. Nucleic Acids Res. 1984 Feb 24;12(4):1889–1900. doi: 10.1093/nar/12.4.1889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Peattie D. A. Direct chemical method for sequencing RNA. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1760–1764. doi: 10.1073/pnas.76.4.1760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schinkel A. H., Groot Koerkamp M. J., Stuiver M. H., Van der Horst G. T., Tabak H. F. Effect of point mutations on in vitro transcription from the promoter for the large ribosomal RNA gene of yeast mitochondria. Nucleic Acids Res. 1987 Jul 24;15(14):5597–5612. doi: 10.1093/nar/15.14.5597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schinkel A. H., Groot Koerkamp M. J., Tabak H. F. Mitochondrial RNA polymerase of Saccharomyces cerevisiae: composition and mechanism of promoter recognition. EMBO J. 1988 Oct;7(10):3255–3262. doi: 10.1002/j.1460-2075.1988.tb03192.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schinkel A. H., Groot Koerkamp M. J., Van der Horst G. T., Touw E. P., Osinga K. A., Van der Bliek A. M., Veeneman G. H., Van Boom J. H., Tabak H. F. Characterization of the promoter of the large ribosomal RNA gene in yeast mitochondria and separation of mitochondrial RNA polymerase into two different functional components. EMBO J. 1986 May;5(5):1041–1047. doi: 10.1002/j.1460-2075.1986.tb04320.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schinkel A. H., Koerkamp M. J., Touw E. P., Tabak H. F. Specificity factor of yeast mitochondrial RNA polymerase. Purification and interaction with core RNA polymerase. J Biol Chem. 1987 Sep 15;262(26):12785–12791. [PubMed] [Google Scholar]
  31. Schinkel A. H., Tabak H. F. Mitochondrial RNA polymerase: dual role in transcription and replication. Trends Genet. 1989 May;5(5):149–154. doi: 10.1016/0168-9525(89)90056-5. [DOI] [PubMed] [Google Scholar]
  32. Sederoff R. R. Structural variation in mitochondrial DNA. Adv Genet. 1984;22:1–108. doi: 10.1016/s0065-2660(08)60038-3. [DOI] [PubMed] [Google Scholar]
  33. Shuey D. J., Attardi G. Characterization of an RNA polymerase activity from HeLa cell mitochondria, which initiates transcription at the heavy strand rRNA promoter and the light strand promoter in human mitochondrial DNA. J Biol Chem. 1985 Feb 10;260(3):1952–1958. [PubMed] [Google Scholar]
  34. Topper J. N., Clayton D. A. Identification of transcriptional regulatory elements in human mitochondrial DNA by linker substitution analysis. Mol Cell Biol. 1989 Mar;9(3):1200–1211. doi: 10.1128/mcb.9.3.1200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tzagoloff A., Myers A. M. Genetics of mitochondrial biogenesis. Annu Rev Biochem. 1986;55:249–285. doi: 10.1146/annurev.bi.55.070186.001341. [DOI] [PubMed] [Google Scholar]
  36. Walberg M. W., Clayton D. A. In vitro transcription of human mitochondrial DNA. Identification of specific light strand transcripts from the displacement loop region. J Biol Chem. 1983 Jan 25;258(2):1268–1275. [PubMed] [Google Scholar]
  37. Ward B. L., Anderson R. S., Bendich A. J. The mitochondrial genome is large and variable in a family of plants (cucurbitaceae). Cell. 1981 Sep;25(3):793–803. doi: 10.1016/0092-8674(81)90187-2. [DOI] [PubMed] [Google Scholar]
  38. Wettstein-Edwards J., Ticho B. S., Martin N. C., Najarian D., Getz G. S. In vitro transcription and promoter strength analysis of five mitochondrial tRNA promoters in yeast. J Biol Chem. 1986 Feb 25;261(6):2905–2911. [PubMed] [Google Scholar]
  39. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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