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Beschreibung:
Translating the information encoded in messenger RNAs (mRNAs) into functional proteins is an essential cellular process carried out by large molecular machines termed ribosomes. Ribosomes are large ribonucleoprotein (RNP) particles, whose biogenesis is an energetically demanding and highly regulated process. During the early stages of ribosome biogenesis, the ribosomal RNAs (rRNAs) get covalently modified. One of the most abundant of these covalent modifications is the methylation of the 2’ hydroxyl group of the ribose (2’-O-Me) in specific nucleotides of the rRNA. Many of these 2’-O-methylated sites are located in functionally important regions of the matured ribosome, such as the peptidyl transferase center (PTC) or the decoding center. Unsurprisingly, aberrations in 2’-O-Me are associated with pathological developments such as cancer and neurological diseases in human. In archaea and eukaryotes 2’-O-Me modifications on rRNA are transferred by the Box C/D enzymes, which are multi-component RNPs that use guide RNAs to mediate site specific 2’-O-methylation on rRNA. Most of the available structural and functional data on the Box C/D RNP enzymes are based on the archaeal enzyme. Conversely, only little is known on the structural and functional details of the eukaryotic Box C/D enzyme. Therefore, the archaeal system is being used as a structural and functional proxy for the eukaryotic enzyme. To expand to structural and functional knowledge about the eukaryotic Box C/D small nucleolar ribonucleoprotein (snoRNP) enzymes and examine the validity of the archaeal enzymes as a proxy I used a combination of biochemical, analytical and structural methods to analyze and characterize two subcomplexes of the eukaryotic Box C/D snoRNP from S. cerevisiae in vitro. Using fluorescence-based electrophoretic mobility shift assays I could characterize the binding requirements and affinities between the eukaryotic and archaeal Box C/D primary RNA-binding protein Snu13 and L7Ae, respectively, and the lesser conserved of two protein ...