Protein synthesis in Archaea. mechanism of dissociation from ribosomes of the conserved translation factor aIF6; development of an in vitro transcription/translation system from the thermophilic archaeon Sulfolobus solfataricus

This work describes two experimental studies performed for my doctoral thesis. The general subject is the analysis of the features and mechanisms of protein synthesis in extremely thermophilic Archaea. The subject is interesting and relevant from different points of view. Firstly, it has long been known that archaeal translation shares several features and components with the corresponding eukaryal process. Investigating the common themes between archaeal and eukaryal protein synthesis may help to shed light on the evolutionary origin thereof and to reconstruct the pathways whereby Eukarya emerged from the common tree of life. Secondly, extremely thermophilic Archaea have a lot of potential for biotechnological development, for instance as a source of thermostable enzymes to be used for both medical and industrial purposes. In the first part of my work, I shall describe a functional analysis of the archaeal translation factor aIF6, a protein shared by the Archaea and the Eukarya but not found in Bacteria. Although the eukaryal factor (eIF6) has been studied extensively, its function in translation is still imperfectly understood. It is established that it acts as a ribosome anti-association factor, binding to the large subunit and preventing its premature joining with the small subunit. To allow the 60S ribosome to enter the translation cycle, eIF6 must be actively released, a process that depends on the action of another factor, Sdo1/SBDS, which is also found in Archaea, and of a GTPase, EFL1, that instead does not have an archaeal homologue. In my work, I studied the mechanism of aIF6 release from archaeal large ribosomal subunits, using as the model organism the thermophilic archaeon Sulfolobus solfataricus. The results I obtained show that detachment of aIF6 from ribosomes requires the GTPase activity of elongation factor 2 (aEF2), while the Sdo1/SBDS homologue apparently inhibits rather than promoting aIF6 release. The function of archaeal Sdo1 remains therefore to be understood and must be studied further. In the second part of work, I developed a coupled in vitro transcription/translation system for cell-free protein synthesis from cell lysates of S. solfataricus. The essential element in this expression system is a strong promoter derivedfrom 16S/23S rRNA-encoding DNA promoter from the archaebacterium Sulfolobus sp. P2 that produces, with high efficiency, specific mRNAs. I show that this method permits the efficient synthesis in vitro at high temperature of biologically active proteins.