Characterisation of the eukaryotic ribosome biogenesis factors, Nob1, Dim2, and Tsr1, and their interactions with RNA
McCaughan, Urszula Maria
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Ribosome biosynthesis in eukaryotes is a complex process involving over 200 accessory factors. Nob1, Dim2, and Tsr1 are three conserved factors that are all involved in the late processing steps of the small subunit (40S) pre-rRNA. Depletion of any of these factors leads to the accumulation of the immature 20S pre-rRNA. Nob1, an essential protein in yeast, performs the final cleavage of small subunit rRNA giving rise to the mature particle. It is aided in this process by other proteins such as Dim2. Previously, the two proteins have been shown to interact. Nob1 function was found to be more efficient in the presence of Dim2. Previous studies also indicated that Nob1 binds a site on the pre-40S that is distal to the cleavage site while Dim2 binds proximally. Using analytical gel filtration, electrophoretic mobility shift assays, and isothermal titration calorimetry we show that Nob1 does not interact with the distal binding site in vitro. Instead, a stable complex with a micromolar disassociation constant can be formed with a sequence derived from the cleavage site. Thus, Nob1 and Dim2 appear to be competing for this site. The interaction with both proteins is blocked when this sequence is sequestered in a hairpin structure, which has been previously predicted to form at this site. By altering individual bases in the RNA sequence, we have identified the sequence determinants for Nob1-rRNA recognition. Tsr1 function is unknown to date. It shares sequence similarity with certain GTPases; however, no GTP binding has been identified in previous studies. The depletion of this factor leads to a similar phenotype as the depletion of Nob1 and Dim2. By screening various deletion constructs, we have obtained good quality, diffracting crystals of yeast Tsr1. However, due to time constraints, the full structure has not been solved. Here we present the initial analysis of the crystallographic data and the potential for solving the structure in the future. Overall, the data presented in this thesis bring insight into the final step of small subunit ribosome maturation.