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. 1987 Jul 24;15(14):5597–5612. doi: 10.1093/nar/15.14.5597

Effect of point mutations on in vitro transcription from the promoter for the large ribosomal RNA gene of yeast mitochondria.

A H Schinkel, M J Groot Koerkamp, M H Stuiver, G T Van der Horst, H F Tabak
PMCID: PMC306009  PMID: 3302943

Abstract

Initiation of transcription on mitochondrial DNA of Saccharomyces cerevisiae was studied in an in vitro system with a mtRNA polymerase fraction reconstituted from separately purified components and with DNA templates containing the promoter of the gene coding for large rRNA. The effect of various point mutations in this promoter region was quantitated in assays containing a wildtype promoter in equimolar amount as internal control. Despite the strong conservation around the position at which RNA initiation occurs (ATATAAGTApuTA, initiation nucleotide underlined), none of the single point mutations abolished transcription-initiation completely. Some reduce the efficiency of initiation to 10-20% compared to the wild type promoter, while others have a much less pronounced effect. A change of the A at position +4 into a G even results in a promoter up mutation. Remarkably, alteration of the A at position +1 into a G or a T affects the efficiency of initiation only slightly and initiation is maintained at the same position.

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Selected References

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

  1. 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]
  2. Biswas T. K., Getz G. S. A critical base in the yeast mitochondrial nonanucleotide promoter. Abolition of promoter activity by mutation at the -2 position. J Biol Chem. 1986 Mar 25;261(9):3927–3930. [PubMed] [Google Scholar]
  3. 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]
  4. Christianson T., Rabinowitz M. Identification of multiple transcriptional initiation sites on the yeast mitochondrial genome by in vitro capping with guanylyltransferase. J Biol Chem. 1983 Nov 25;258(22):14025–14033. [PubMed] [Google Scholar]
  5. Colin Y., Baldacci G., Bernardi G. A new putative gene in the mitochondrial genome of Saccharomyces cerevisiae. Gene. 1985;36(1-2):1–13. doi: 10.1016/0378-1119(85)90064-2. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Kammerer W., Deuschle U., Gentz R., Bujard H. Functional dissection of Escherichia coli promoters: information in the transcribed region is involved in late steps of the overall process. EMBO J. 1986 Nov;5(11):2995–3000. doi: 10.1002/j.1460-2075.1986.tb04597.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Norris K., Norris F., Christiansen L., Fiil N. Efficient site-directed mutagenesis by simultaneous use of two primers. Nucleic Acids Res. 1983 Aug 11;11(15):5103–5112. doi: 10.1093/nar/11.15.5103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Osinga K. A., De Haan M., Christianson T., Tabak H. F. A nonanucleotide sequence involved in promotion of ribosomal RNA synthesis and RNA priming of DNA replication in yeast mitochondria. Nucleic Acids Res. 1982 Dec 20;10(24):7993–8006. doi: 10.1093/nar/10.24.7993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Osinga K. A., Van der Bliek A. M., Van der Horst G., Groot Koerkamp M. J., Tabak H. F., Veeneman G. H., Van Boom J. H. In vitro site-directed mutagenesis with synthetic DNA oligonucleotides yields unexpected deletions and insertions at high frequency. Nucleic Acids Res. 1983 Dec 20;11(24):8595–8608. doi: 10.1093/nar/11.24.8595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Sakonju S., Bogenhagen D. F., Brown D. D. A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5' border of the region. Cell. 1980 Jan;19(1):13–25. doi: 10.1016/0092-8674(80)90384-0. [DOI] [PubMed] [Google Scholar]
  14. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Sims P. F., Minter S. J., Stancombe R., Gent M. E., Andrews J., Waring R. B., Towner P., Davies R. W. A modified two primer approach to oligonucleotide-directed in vitro mutagenesis. Biochimie. 1985 Jul-Aug;67(7-8):841–847. doi: 10.1016/s0300-9084(85)80177-2. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]

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