Sulfolobus solfataricus: A model system to study control of gene expression in Archaea

Sulfolobus solfataricus is an archaeal organism, growing at pH 3 and 80°C. Archaea have a basal transcription apparatus resembling the eukaryotic one. In this thesis regulated transcription in archaea is studied. Little is known about this process in archea and due to the ease of cultivation Sulfolobus is an optimal system for its study.; In Sulfolobus, two copies of TFB are present. TFB is a homolog of the eukaryotic transcription factor II B (TFIIB). The tbf-2 mRNA level varies depending on the conditions of growth (present in exponential phase, absent in stationary phase) and after heat shock (absent after heat shock). In contrast, the tfb-1 mRNA is always present.; A method for the measurement of mRNA stability, employing actinomycin D, was developed. Using this method the half-lives of tfb-1 mRNA (>120 min) and tfb-2 mRNA (18 min) were determined. This method indicates that different mRNA stability has a role in the differential expression of tfb-1 and tfb-2.; The regulation of genes involved in carbohydrate metabolism on sucrose (inducing) and on sucrose supplemented with yeast extract (repressing) was studied. The most affected gene was lacS, coding for the beta-glycosidase LacS. A 19-fold difference in enzyme activity between the two conditions was found. LacS- mutants, called car from catabolite repression, showed a reduced activity and a lowered response to the type of carbon source. The regulation of LacS was previously demonstrated to occur at the level of transcription. In order to determine whether the expression of lacS occurred at the level of rate of transcription or mRNA stability, the half-life of lacS mRNA under inducing and repressing conditions were measured. Identical half-life values (10 min) were found in both cases, therefore control at the level of mRNA stability was excluded. The stability of lacS mRNA in the car1 mutant was 7 min. It is concluded that the car locus produces a factor controlling regulation and at the same time determines the altered car mutant phenotype.