Regulation of archaeal gene expression by basal transcription factors variants

The majority of cell types contain the same genetic material to encode the various cellular components. Differences in cell types (e.g. neurons as compared to muscle cells) arise because different parts of the genetic material are read out. RNA polymerises (RNAPs) are a crucial part of the cellular machinery involved in the selective reading mechanism and it is therefore important to understand their molecular structure and function in considerable detail. RNAPs can, however, not carry out this function on their own ¿ they rely on the assistance of basal factors that help them to bind to the right places of the genetic material. We are using a class of simple microorganisms (archaea) that, amazingly, contain an RNAP and basal factors that are very similar to those found in higher organisms, including humans. By studying the biochemical properties of these archaeal components we hope to gain a better understanding of the function of basal factors and how they guide RNAPs to the correct starts sites. An additional advantage of the chosen archaeal model system is that these archaea grow under biologically extremely hostile conditions (in salt-saturated lakes bombarded with high intensity sunlight). By studying organisms that are capable of withstanding such environmental challenges we also gain insights how living systems could evolve and function on other planets and thus provide the basis for understanding extraterrestrial life forms.

Archaea represent one of the three evolutionary domains of life. They are prokaryotes resembling some of the earliest known form of life on earth and are nowadays found in a large variety of ecological niches that include normal mesophilic environments, but also environments that are too hostile to support any representatives of the two other evolutionary domains (i.e. bacteria and eukaryotes). The adaptation of archaea to life under extreme conditions (e.g. low and high temperature; extreme acidity and alkalinity; environments containing high salt concentrations) is of great biological, biochemical and biophysical interest. Archaea have gained additional significance because of their macromolecular information processing machinery (DNA replication, transcription, repair and protein translation) closely resembles the core machineries found in eukaryotic cells. They contain a RNA polymerase (RNAP) that is directly comparable to eukaryotic RNAPIIs in terms of subunit composition, primary amino acid sequence and the quaternary arrangement of subunits within the intact enzyme. In addition, archaeal promoters have TATA box-like sequence elements that are specifically recognised by archaeal homologs of TATA-binding protein (TBP), TFB (homolog of eukaryotic TFIIB) and TFE (a homolog of eukaryotic TFIIE). The recent analysis of the Halobacterium genome, an archaeon growing under extreme halophilic conditions, has revealed the existence of six TBP and seven TFB variants that are likely to display different functional properties. This may be a highly effective strategy for controlling differential gene expression because the TBP/TFB variants are likely to differ in efficiency and specificity by which archaeal core promoter elements (TATA-boxes, and B-recognition elements [BREs]) are recognised. For the proposed project we will clone, express and purify the Halobacterium TBP and TFB variants as recombinant proteins and determine their optimal target sequences using binding-site selection assays (SELEX) under specially developed halophilic conditions. A full characterisation of the functions of Halobacterium basal factor variants will greatly benefit from the availability of an in vitro transcription system capable of carrying out promoter-specific transcription under extreme halophilic conditions (i.e. in the presence of 2-5M KCI). Such a system does not currently exist, but preliminary work has provided us with clues how this may be achieved. We will attempt to develop a (partial) purification procedure for purifying endogenous Halobacterium RNAP capable of functioning under extreme halophilic conditions. The proposed experimental approach will complement currently existing genetic tools for studying gene expression in extreme halophiles and will make a substantial contribution to the systems biology of this organism.