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 language||English (US)|
|State||Published - 1997|
ASJC Scopus subject areas
- Molecular Biology