Biochemical characterization of archaeal transcription factors

G. M. Coldn Gonzalez, L. K. McNeil, C. I. Reich, G. J. Olsen

Research output: Contribution to journalArticle

Abstract

Archaea and Eucarya share similarities in the RNA polymerase subunits and protein factors involved in transcription initiation. However, unlike the Eukarya, there is a single RNA polymerase in the Archaea. Archaeal homologs of the basai transcription factors TFIIB (TFB) and TATA-binding protein (TBP) have been cloned and expressed. We are examining the mode of action of these factors from both thermophilic and mesophilic species. We are particularly interested in two facets of transcription initiation: the differences between high and moderate temperature versions of this system, and the differences between the three classes of promoters that are handled by distinct polymerases and transcription factors in eukaryotes. We have amplified the genes for TBP from the thermophile Methanococcus jannaschii and the mesophiles Methanococcus maripaludis and Methanoplanus limicola; and the TFB gene from the thermophile Thermococcus litoralis and the mesophiles Mp. limicola and Me. maripaludis. These genes were cloned into appropriate vectors for expression and purification of the recombinant proteins in E coll. These transcription factors are being assayed for their abilities to bind different classes of promoters by gel mobility shift assays and fluorescence anisotropy. Initial results show binding of Mp. limicola TBP to a tRNA promoter, while Me. maripaludis and Me. jannashii TBP do not exhibit detectable binding under the same gel mobility shift assay conditions. In all cases increased binding is observed when T. litoralis TFB is added to the reaction. Binding is enhanced by increasing the total salt concentration to 280 mM in the ONA mobility shift assay reactions. The TFB from the thermophile T. litoralis stabilizes the binding of mesophilic and thermophilic TATA-binding proteins. Both thermophilic and mesophilic proteins bind optimaly at higher ionic strenght conditions than those reported for eukaryotic homologs. A comparative approach of binding kinetics towards different types of promoters as well as mutational analyses will provide insights into basal gene expression in the archaeal domain of life.

Original languageEnglish (US)
Pages (from-to)A943
JournalFASEB Journal
Volume11
Issue number9
StatePublished - Dec 1 1997

Fingerprint

TATA-Box Binding Protein
Transcription Factor TFIIB
thermophilic microorganisms
binding proteins
Transcription Factors
transcription factors
Electrophoretic Mobility Shift Assay
Eukaryota
promoter regions
Assays
Genes
Archaea
DNA-Directed RNA Polymerases
Transcription
DNA-directed RNA polymerase
Methanomicrobiaceae
assays
Thermococcus
Methanococcus
Methanocaldococcus

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

Cite this

Coldn Gonzalez, G. M., McNeil, L. K., Reich, C. I., & Olsen, G. J. (1997). Biochemical characterization of archaeal transcription factors. FASEB Journal, 11(9), A943.

Biochemical characterization of archaeal transcription factors. / Coldn Gonzalez, G. M.; McNeil, L. K.; Reich, C. I.; Olsen, G. J.

In: FASEB Journal, Vol. 11, No. 9, 01.12.1997, p. A943.

Research output: Contribution to journalArticle

Coldn Gonzalez, GM, McNeil, LK, Reich, CI & Olsen, GJ 1997, 'Biochemical characterization of archaeal transcription factors', FASEB Journal, vol. 11, no. 9, pp. A943.
Coldn Gonzalez GM, McNeil LK, Reich CI, Olsen GJ. Biochemical characterization of archaeal transcription factors. FASEB Journal. 1997 Dec 1;11(9):A943.
Coldn Gonzalez, G. M. ; McNeil, L. K. ; Reich, C. I. ; Olsen, G. J. / Biochemical characterization of archaeal transcription factors. In: FASEB Journal. 1997 ; Vol. 11, No. 9. pp. A943.
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