Regulation of transcription in hyperthermophilic archaea

The aim of the research presented here was to insight in the mechanisms by which transcription in hyperthermophilic archaea is regulated. To accomplish this, we have aimed (I) to identify transcriptional regulatory proteins from hyperthermophilic archaea, (II) to characterize these proteins, and (III) to determine how these proteins modulate the process of transcription initiation.Chapter 1 describes the characteristics of the archaeal transcription machinery, and compiles transcription-related data that was obtained in the past two decades. The archaeal transcription machinery appears to be a simplified version of the eukaryal RNA polymerase II system, lacking various general transcription factors that are essential for eukaryal transcription initiation. However, archaeal genomes encode TFE, a homologue eukaryal TFIIEatranscription factor. Its stimulatory role in transcription is described in Chapter 2 . Although the archaeal transcription machinery is eukaryal-like, many genes encoding members of bacterial regulatory protein families can be found within archaeal genomes. Members of the Lrp family are most abundantly present in archaea and Chapter 3 describes the properties of Lrp-like proteins. When this research project was started, fully sequenced archaeal genomes just became available . Only the gene encoding LrpA from P. furiosus had previously been identified in our laboratory and by others , and our initial strategy included the characterization of this protein, which is described in Chapter 4 . LrpA was shown to negatively autoregulate its own transcription in a ligand-independent manner. The efficient production and purification of recombinant LrpA enabled crystallization of the protein and Chapter 5 describes its resolved three-dimensional structure, which is the first structure of a member of the Lrp family. Subsequently, during the participation of our laboratory in the S.solfataricus P2 genome sequencing project, we were able to identify candidate regulatory genes in a more directed, bioinformatics-based approach, resulting in the identification and characterization of LysM and ChoR. LysM is another example of an archaeal Lrp-like protein, and in Chapter 6 we have used the genomic context of LysM in the S.solfataricus genome to experimentally identify its physiological target and ligand. This study indicates for the first time that an Lrp-like protein may activate archaeal transcriptional.Besides bacterial-like regulators, archaeal genomes encode unique archaeal-specific regulators that can be identified on the basis of a present DNA-binding domain. A putative regulator for c opper ho meostasis (ChoR) was identified in the S.solfataricus genome on the basis of a predicted HTH DNA-binding domain and a metal-binding domain. In Chapter 7 it is demonstrated that ChoR is indeed a metal-responsive DNA-binding protein that is most likely involved in the repression of a heavy metal-efflux system.Chapter 8 summarizes the data presented in this thesis, and adds some concluding remarks with respect to the implications of the work