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. 1988 Jul;8(7):2910–2916. doi: 10.1128/mcb.8.7.2910

Purification of Xenopus laevis mitochondrial RNA polymerase and identification of a dissociable factor required for specific transcription.

D F Bogenhagen 1, N F Insdorf 1
PMCID: PMC363510  PMID: 2457154

Abstract

The Xenopus laevis mitochondrial RNA (mtRNA) polymerase was purified to near homogeneity with an overall yield approaching 50%. The major polypeptides in the final fraction were a doublet of proteins of approximately 140 kilodaltons that copurified with the mtRNA polymerase activity. It appeared likely that the smaller polypeptide is a breakdown product of the larger one. The highly purified polymerase was active in nonspecific transcription but required a dissociable factor for specific transcription of X. laevis mtDNA. The factor could be resolved from mtRNA polymerase by hydrophobic chromatography and had a sedimentation coefficient of 3.0 S. The transcription factor eluted from both the hydrophobic column and a Mono Q anion-exchange column as a single symmetrical peak. The mtRNA polymerase and this factor together are necessary and sufficient for active transcription from four promoters located in a noncoding region of the mtDNA genome between the gene for tRNA(Phe) and the displacement loop.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson S., Bankier A. T., Barrell B. G., de Bruijn M. H., Coulson A. R., Drouin J., Eperon I. C., Nierlich D. P., Roe B. A., Sanger F. Sequence and organization of the human mitochondrial genome. Nature. 1981 Apr 9;290(5806):457–465. doi: 10.1038/290457a0. [DOI] [PubMed] [Google Scholar]
  2. Biswas T. K., Edwards J. C., Rabinowitz M., Getz G. S. Characterization of a yeast mitochondrial promoter by deletion mutagenesis. Proc Natl Acad Sci U S A. 1985 Apr;82(7):1954–1958. doi: 10.1073/pnas.82.7.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Bogenhagen D. F., Romanelli M. F. Template sequences required for transcription of Xenopus laevis mitochondrial DNA from two bidirectional promoters. Mol Cell Biol. 1988 Jul;8(7):2917–2924. doi: 10.1128/mcb.8.7.2917. [DOI] [PMC free article] [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. Cairns S. S., Bogenhagen D. F. Mapping of the displacement loop within the nucleotide sequence of Xenopus laevis mitochondrial DNA. J Biol Chem. 1986 Jun 25;261(18):8481–8487. [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. Chomyn A., Mariottini P., Cleeter M. W., Ragan C. I., Matsuno-Yagi A., Hatefi Y., Doolittle R. F., Attardi G. Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory-chain NADH dehydrogenase. Nature. 1985 Apr 18;314(6012):592–597. doi: 10.1038/314592a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Kelly J. L., Greenleaf A. L., Lehman I. R. Isolation of the nuclear gene encoding a subunit of the yeast mitochondrial RNA polymerase. J Biol Chem. 1986 Aug 5;261(22):10348–10351. [PubMed] [Google Scholar]
  14. Kelly J. L., Lehman I. R. Yeast mitochondrial RNA polymerase. Purification and properties of the catalytic subunit. J Biol Chem. 1986 Aug 5;261(22):10340–10347. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Levens D., Howley P. M. Novel method for identifying sequence-specific DNA-binding proteins. Mol Cell Biol. 1985 Sep;5(9):2307–2315. doi: 10.1128/mcb.5.9.2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Levens D., Lustig A., Rabinowitz M. Purification of mitochondrial RNA polymerase from Saccharomyces cerevisiae. J Biol Chem. 1981 Feb 10;256(3):1474–1481. [PubMed] [Google Scholar]
  18. Masters B. S., Stohl L. L., Clayton D. A. Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. Cell. 1987 Oct 9;51(1):89–99. doi: 10.1016/0092-8674(87)90013-4. [DOI] [PubMed] [Google Scholar]
  19. Reid B. D., Parsons P. Partial purification of mitochondrial RNA polymerase from rat liver. Proc Natl Acad Sci U S A. 1971 Nov;68(11):2830–2834. doi: 10.1073/pnas.68.11.2830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Shaltiel S. Hydrophobic chromatography. Methods Enzymol. 1974;34:126–140. doi: 10.1016/s0076-6879(74)34012-8. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Winkley C. S., Keller M. J., Jaehning J. A. A multicomponent mitochondrial RNA polymerase from Saccharomyces cerevisiae. J Biol Chem. 1985 Nov 15;260(26):14214–14223. [PubMed] [Google Scholar]
  25. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]
  26. Wu G. J., Dawid I. B. Purification and properties of mitochondrial deoxyribonucleic acid dependent ribonucleic acid polymerase from ovaries of Xenopus laevis. Biochemistry. 1972 Sep 12;11(19):3589–3595. doi: 10.1021/bi00769a015. [DOI] [PubMed] [Google Scholar]

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