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. 1999 Aug;152(4):1325–1333. doi: 10.1093/genetics/152.4.1325

Cell-free transcription at 95 degrees: thermostability of transcriptional components and DNA topology requirements of Pyrococcus transcription.

C Hethke 1, A Bergerat 1, W Hausner 1, P Forterre 1, M Thomm 1
PMCID: PMC1460703  PMID: 10430563

Abstract

Cell-free transcription of archaeal promoters is mediated by two archaeal transcription factors, aTBP and TFB, which are orthologues of the eukaryotic transcription factors TBP and TFIIB. Using the cell-free transcription system described for the hyperthermophilic Archaeon Pyrococcus furiosus by Hethke et al., the temperature limits and template topology requirements of archaeal transcription were investigated. aTBP activity was not affected after incubation for 1 hr at 100 degrees. In contrast, the half-life of RNA polymerase activity was 23 min and that of TFB activity was 3 min. The half-life of a 328-nt RNA product was 10 min at 100 degrees. Best stability of RNA was observed at pH 6, at 400 mm K-glutamate in the absence of Mg(2+) ions. Physiological concentrations of K-glutamate were found to stabilize protein components in addition, indicating that salt is an important extrinsic factor contributing to thermostability. Both RNA and proteins were stabilized by the osmolyte betaine at a concentration of 1 m. The highest activity for RNA synthesis at 95 degrees was obtained in the presence of 1 m betaine and 400 mm K-glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70 degrees and 90 degrees. However, negatively supercoiled DNA was the preferred template at all temperatures tested. Analyses of transcripts from plasmid topoisomers harboring the glutamate dehydrogenase promoter and of transcription reactions conducted in the presence of reverse gyrase indicate that positive supercoiling of DNA inhibits transcription from this promoter.

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Selected References

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  1. Adams M. W. Enzymes and proteins from organisms that grow near and above 100 degrees C. Annu Rev Microbiol. 1993;47:627–658. doi: 10.1146/annurev.mi.47.100193.003211. [DOI] [PubMed] [Google Scholar]
  2. Bell S. D., Jaxel C., Nadal M., Kosa P. F., Jackson S. P. Temperature, template topology, and factor requirements of archaeal transcription. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15218–15222. doi: 10.1073/pnas.95.26.15218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bird T., Burbulys D., Wu J. J., Strauch M. A., Hoch J. A., Spiegelman G. B. The effect of supercoiling on the in vitro transcription of the spoIIA operon from Bacillus subtilis. Biochimie. 1992 Jul-Aug;74(7-8):627–634. doi: 10.1016/0300-9084(92)90134-z. [DOI] [PubMed] [Google Scholar]
  4. Charbonnier F., Erauso G., Barbeyron T., Prieur D., Forterre P. Evidence that a plasmid from a hyperthermophilic archaebacterium is relaxed at physiological temperatures. J Bacteriol. 1992 Oct;174(19):6103–6108. doi: 10.1128/jb.174.19.6103-6108.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Dvir A., Garrett K. P., Chalut C., Egly J. M., Conaway J. W., Conaway R. C. A role for ATP and TFIIH in activation of the RNA polymerase II preinitiation complex prior to transcription initiation. J Biol Chem. 1996 Mar 29;271(13):7245–7248. doi: 10.1074/jbc.271.13.7245. [DOI] [PubMed] [Google Scholar]
  6. Forterre P., Confalonieri F., Charbonnier F., Duguet M. Speculations on the origin of life and thermophily: review of available information on reverse gyrase suggests that hyperthermophilic procaryotes are not so primitive. Orig Life Evol Biosph. 1995 Jun;25(1-3):235–249. doi: 10.1007/BF01581587. [DOI] [PubMed] [Google Scholar]
  7. Gartenberg M. R., Wang J. C. Positive supercoiling of DNA greatly diminishes mRNA synthesis in yeast. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11461–11465. doi: 10.1073/pnas.89.23.11461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hausner W., Thomm M. Purification and characterization of a general transcription factor, aTFB, from the archaeon Methanococcus thermolithotrophicus. J Biol Chem. 1993 Nov 15;268(32):24047–24052. [PubMed] [Google Scholar]
  9. Hausner W., Wettach J., Hethke C., Thomm M. Two transcription factors related with the eucaryal transcription factors TATA-binding protein and transcription factor IIB direct promoter recognition by an archaeal RNA polymerase. J Biol Chem. 1996 Nov 22;271(47):30144–30148. doi: 10.1074/jbc.271.47.30144. [DOI] [PubMed] [Google Scholar]
  10. Herzel H., Weiss O., Trifonov E. N. Sequence periodicity in complete genomes of archaea suggests positive supercoiling. J Biomol Struct Dyn. 1998 Oct;16(2):341–345. doi: 10.1080/07391102.1998.10508251. [DOI] [PubMed] [Google Scholar]
  11. Hethke C., Geerling A. C., Hausner W., de Vos W. M., Thomm M. A cell-free transcription system for the hyperthermophilic archaeon Pyrococcus furiosus. Nucleic Acids Res. 1996 Jun 15;24(12):2369–2376. doi: 10.1093/nar/24.12.2369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hüdepohl U., Reiter W. D., Zillig W. In vitro transcription of two rRNA genes of the archaebacterium Sulfolobus sp. B12 indicates a factor requirement for specific initiation. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5851–5855. doi: 10.1073/pnas.87.15.5851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kengen S. W., Luesink E. J., Stams A. J., Zehnder A. J. Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur J Biochem. 1993 Apr 1;213(1):305–312. doi: 10.1111/j.1432-1033.1993.tb17763.x. [DOI] [PubMed] [Google Scholar]
  14. Kugel J. F., Goodrich J. A. Promoter escape limits the rate of RNA polymerase II transcription and is enhanced by TFIIE, TFIIH, and ATP on negatively supercoiled DNA. Proc Natl Acad Sci U S A. 1998 Aug 4;95(16):9232–9237. doi: 10.1073/pnas.95.16.9232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lindahl T. Irreversible heat inactivation of transfer ribonucleic acids. J Biol Chem. 1967 Apr 25;242(8):1970–1973. [PubMed] [Google Scholar]
  16. López-García P., Forterre P. DNA topology in hyperthermophilic archaea: reference states and their variation with growth phase, growth temperature, and temperature stresses. Mol Microbiol. 1997 Mar;23(6):1267–1279. doi: 10.1046/j.1365-2958.1997.3051668.x. [DOI] [PubMed] [Google Scholar]
  17. Marguet E., Forterre P. DNA stability at temperatures typical for hyperthermophiles. Nucleic Acids Res. 1994 May 11;22(9):1681–1686. doi: 10.1093/nar/22.9.1681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marsh T. L., Reich C. I., Whitelock R. B., Olsen G. J. Transcription factor IID in the Archaea: sequences in the Thermococcus celer genome would encode a product closely related to the TATA-binding protein of eukaryotes. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4180–4184. doi: 10.1073/pnas.91.10.4180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meier T., Schickor P., Wedel A., Cellai L., Heumann H. In vitro transcription close to the melting point of DNA: analysis of Thermotoga maritima RNA polymerase-promoter complexes at 75 degrees C using chemical probes. Nucleic Acids Res. 1995 Mar 25;23(6):988–994. doi: 10.1093/nar/23.6.988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nikolov D. B., Chen H., Halay E. D., Usheva A. A., Hisatake K., Lee D. K., Roeder R. G., Burley S. K. Crystal structure of a TFIIB-TBP-TATA-element ternary complex. Nature. 1995 Sep 14;377(6545):119–128. doi: 10.1038/377119a0. [DOI] [PubMed] [Google Scholar]
  21. Ouzounis C., Sander C. TFIIB, an evolutionary link between the transcription machineries of archaebacteria and eukaryotes. Cell. 1992 Oct 16;71(2):189–190. doi: 10.1016/0092-8674(92)90347-f. [DOI] [PubMed] [Google Scholar]
  22. Phipps B. M., Hoffmann A., Stetter K. O., Baumeister W. A novel ATPase complex selectively accumulated upon heat shock is a major cellular component of thermophilic archaebacteria. EMBO J. 1991 Jul;10(7):1711–1722. doi: 10.1002/j.1460-2075.1991.tb07695.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Roovers M., Hethke C., Legrain C., Thomm M., Glansdorff N. Isolation of the gene encoding Pyrococcus furiosus ornithine carbamoyltransferase and study of its expression profile in vivo and in vitro. Eur J Biochem. 1997 Aug 1;247(3):1038–1045. doi: 10.1111/j.1432-1033.1997.01038.x. [DOI] [PubMed] [Google Scholar]
  24. Rowlands T., Baumann P., Jackson S. P. The TATA-binding protein: a general transcription factor in eukaryotes and archaebacteria. Science. 1994 May 27;264(5163):1326–1329. doi: 10.1126/science.8191287. [DOI] [PubMed] [Google Scholar]
  25. Santoro M. M., Liu Y., Khan S. M., Hou L. X., Bolen D. W. Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry. 1992 Jun 16;31(23):5278–5283. doi: 10.1021/bi00138a006. [DOI] [PubMed] [Google Scholar]
  26. Scholz S., Sonnenbichler J., Schäfer W., Hensel R. Di-myo-inositol-1,1'-phosphate: a new inositol phosphate isolated from Pyrococcus woesei. FEBS Lett. 1992 Jul 20;306(2-3):239–242. doi: 10.1016/0014-5793(92)81008-a. [DOI] [PubMed] [Google Scholar]
  27. TETAS M., LOWENSTEIN J. M. The effect of bivalent metal ions on the hydrolysis of adenosine di- and triphosphate. Biochemistry. 1963 Mar-Apr;2:350–357. doi: 10.1021/bi00902a030. [DOI] [PubMed] [Google Scholar]
  28. Tse-Dinh Y. C. Regulation of the Escherichia coli DNA topoisomerase I gene by DNA supercoiling. Nucleic Acids Res. 1985 Jul 11;13(13):4751–4763. doi: 10.1093/nar/13.13.4751. [DOI] [PMC free article] [PubMed] [Google Scholar]

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