The Transcriptional Regulation of the alcohol Dehydrogenase gene in Sulfolobus solfataricus: identification of Regulatory Sequences and binding Factors

INTRODUCTION: The understanding of archaeal transcription initiation has been deepened by recent progress, which include genome sequencing, biochemical approaches, and the development of vector/transformation systems. Although it has been demonstrated both with functional and structural approaches that the basal transcription apparatus of Archaea corresponds to a simplified core machinery of the eukaryal RNA polymerase II system, little is currently known about how regulation of archaeal transcription is achieved. In order to give insight in gene regulation in thermophilic Archaea, we examined the expression of the alcohol dehydrogenase (adh) gene in Sulfolobus solfataricus. MATERIAL AND METHODS: S. solfataricus cultures were grown in different conditions among which DSM182 medium supplemented with 1 mM benzaldehyde, the natural substrate of the ADH enzyme, and harvested at exponential or stationary phase. mRNA and crude extracts were prepared according to conventional procedures to perform Northern blot analysis and enzyme assays, respectively. Radiolabelled DNA fragments for band shift analysis have been prepared by endonuclease restriction of the 300 bp region upstream of the adh gene. Two synthetic single stranded oligonucleotides encompassing two palindromic sequences located in the adh promoter have also been annealed, labelled and used as probe. 15000 cpm of radiolabelled fragments or annealed oligonucleotides were used for each binding reaction. After binding, samples were loaded on a running 5% polyacrylamide gel containing TBE. The gel was dried and exposed to autoradiography. RESULTS: Studies based on Northern analysis and enzyme assays, demonstrated that the adh gene is induced in the early growth phase by addition to the medium of benzaldehyde, the natural ADH substrate. We also detected in the crude extracts of induced cells, factor(s) specifically able to bind to a 5' regulatory sequence located immediately upstream of the TATA box, suggesting that this region is responsible for the regulated expression of its gene. Further molecular characterisation of the adh promoter allowed the identification of a palindromic cis-acting sequence and of its interacting factor(s). The purification to homogeneity of the factor(s) and the elucidation of its role in transcription regulation are underway.