Protein-Protein interactions involved in the biogenesis of eukaryotic small ribosomal subunits
Castle, Cathy Lynn.
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Ribosome biogenesis is a complicated process involving numerous proteins and modification factors. The process has been well-documented in prokaryotic cells where it is much less complex than the process involved in eukaryotic cells. In eukaryotes, much of what is known about ribosome biogenesis has been learned from studies in the yeast Saccharomyces cerevisiae. Far less has been learned about higher eukaryotes such as humans. However, among organisms in all three domains of life, ribosome structure and function is well conserved. The biogenesis of ribosomal subunits is dynamic, complicated, and, in S. cerevisiae, requires over 200 trans-acting factors for synthesis to occur. The focus of this study is synthesis of the small ribosome subunit in eukaryotes. In order to study this process in higher eukaryotes, a mammalian cell culture method was used. This method involves cloning human ribosomal accessory genes using the Gateway system, a rapid and efficient cloning method that permits parallel construction of numerous plasmids in a modular type of system, each containing the desired gene of interest in a variety of vectors. Proteins were selected based on their activity in yeast with emphasis on the final processing step of the small subunit and the proteins that are involved at that step. These proteins are Nob1p, Enp1p, Tsr1p, Rio2p, Rrp20p, Dim1p, and Hrr25p. The corresponding genes were cloned into vectors containing either the coding region for a full-length fluorescent protein or the C-terminus or N-terminus half of the fluorescent protein. The bimolecular fluorescent complementation assay (BiFC) was then utilized to detect protein-protein interactions. Results of this assay demonstrate binary interactions among pairs of this group of proteins and the location within the cell where these interactions take place.