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The biogenesis of the ribosome requires a series of essential modifications of ribosomal RNAs (rRNAs) and their precursor pre-rRNAs. The most abundant of such modifications is the methylation of the ribose 2 ́-OH, which occurs at over 100 rRNA sites in humans. rRNA methylation is known to increase the stability of the ribosome and to be required for accurate and efficient protein translation. While 2’-O methylation sites are known to cluster around the functional centres of the ribosome, the abundance of methylation at each site is known to vary, which may provide a mechanism to fine tune ribosomal function, creating specialized ribosome populations. In eukaryotes and archaea, rRNA 2’-O methylation is mediated by Box C/D ribonucleoprotein particles (RNPs). These particles, referred to as small nucleolar RNPs (snoRNPs) in eukaryotes and small RNPs (sRNPs) in archaea, use a guide RNA in order to direct the methylation of a specific nucleotide on the substrate rRNA. In archaea, each small guide RNA (sRNA) is responsible for the methylation of two rRNA sites using two different separate guide regions. Despite several structures of archaeal Box C/D sRNPs being available, the molecular basis for the regulation of the enzyme and the consequent generation of varying methylation abundances across different rRNA sites remains elusive. In order to understand the mechanism and regulation of the enzyme, I investigated the biochemical properties of archaeal Box C/D sRNPs reconstituted in vitro . Through a combination of biochemical and nuclear magnetic resonance (NMR)-based assays, I could show that archaeal RNPs catalyse the methylation of different substrate rRNA sites with varying degrees of efficiency and cooperativity. Furthermore, using low-resolution small angle scattering (SAS) techniques, I could show that addition of substrate RNAs onto some sRNPs is correlated with the complex undergoing a transition between different oligomeric and/or conformational states, thereby contextualising the multiple sRNP structures observed in previous studies. In the second part of my work, I used a combination of distance restraints derived from NMR and low-resolution information from SAS to obtain the structures of an archaeal sRNP bound to either of its two substrate RNAs by an integrative structural biology approach. As this particle contains flexible regions, the work required the development of a novel algorithm capable of dealing with NMR/SAS signals arising from ensembles, rather than single conformers. Using this tool, I could derive the populations of conformers within ensembles of RNPs bound to different substrate RNAs, which provide a structural basis for the varying methylation efficiency of the enzyme. Ultimately, the work presented here provides a model for understanding one of the mechanism through which specialised ribosome populations are generated in vivo and contributes to the development of novel techniques for integrative structure modelling of flexible systems. |
