Cell-Free Transcription at 95 degree : Thermostability of Transcriptional Components and DNA Topology Requirements of Pyrococcus Transcription

• Published in: Genetics (Austin) Vol. 152; no. 4; pp. 1325 - 1333
• Main Authors: Hethke, C, Bergerat, A, Hausner, W, terre, P, Thomm, M
• Format: Journal Article
• Language: English
• Published: 01.08.1999
• Subjects: Pyrococcus
• ISSN: 0016-6731

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 degree . 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 degree . Best stability of RNA was observed at pH 6, at 400 m M K-glutamate in the absence of Mg super(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 degree was obtained in the presence of 1 M betaine and 400 m M K- glutamate. Positively supercoiled DNA, which was found to exist in Pyrococcus cells, can be transcribed in vitro both at 70 degree and 90 degree . 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.