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Abstract

The thermophilic ascomycete Chaetomium thermophilum gains growing popularity in the field of structural biochemistry and biotechnology due to its thermostable proteome. Recently, this thermophile has been successfully utilized as platform to gain high resolution “snap-shots”, of pre-ribosomal assembly intermediates by cryogenic electron microscopy (cryo-EM). In the model organism Saccharomyces cerevisiae, one of the versatile tools to study ribosome biogenesis is the inducible GAL1-promoter. However, the genetic toolset of the emerging model organism C. thermophilum was limited to the random integration of expression cassettes, allowing to purify pre- ribosomal particles with constitutively expressed biogenesis factors via affinity purification. In this PhD thesis, I focused on the establishment of an inducible promoter system to control dominant negative mutants as new tool to study ribosome biogenesis in the thermophile. Firstly, I addressed an endogenous carbon regulatable expression system that withstands the thermostable requirements. Using single carbon glucose and xylose media enabled me to characterize the transcriptome of C. thermophilum and to screen for the most differentially transcribed genes by mRNA Illumina sequencing. Promoter regions of xylose specifically activated genes were validated in YFP-reporter strains in vivo and biochemically. The most stringently xylose induced promoters originated from a xylosidase-like gene (XYL) and the xylitol-dehydrogenase gene (XDH), which were tested to study the process of ribosome biogenesis. As proof of concept, both promoters allowed us to induce the expression of a well characterized nucleolar biogenesis factor (UTP-6) using the inductive media. Expression of YFP- tagged UTP-6 under control of the XYL-promoter showed clear xylose induction of UTP-6 in vivo. Using the XDH-promoter, FTpA-tagged UTP-6 was overexpressed in xylose grown cells on affinity purified 90S pre-ribosomal particles on Coomassie stained gels. Moreover, the XDH-promoter allowed me to visualize an inducible dominant negative 60S-subunit export defect upon expression of the mutant pre-60S factor rsa4 E117D in a L25-YFP reporter strain. Overall, the provided tools and transcriptomic studies described in my PhD thesis will facilitate the future work with the thermophile and future applications that benefit from its thermostable proteins. I