Eukaryotic ribosome biogenesis, a dynamic and coordinated multistep process which requires more than 150 trans-acting factors, has been intensely studied in the yeast Saccharomyces cerevisiae. This evolutionarily conserved process involves numerous cleavages of pre-rRNA, modification of nucleotides, and concomitant assembly of the ribosomal proteins onto the rRNA. Considerable information is available about the importance of conserved pre-rRNA cleavage events in ribosome biogenesis; however, very little is known about the exact role of modified nucleotides, which cluster within the functionally important regions of the ribosome. One conserved group of modifications is the dimethylation of two adjacent adenosines at the 3´ end of the small subunit rRNA which is ubiquitously carried out by the Dim1/KsgA methyltransferase family. Although dimethylation and KsgA are dispensable for survival in bacteria, the eukaryotic enzyme Dim1 is essential because of its requirement in the early pre-rRNA processing events. Similarly, few other members of the family have also evolved to carryout a second unrelated function in the cell. Almost all of the information about Dim1 was obtained from in vivo experiments in yeast, and has been determined that it is an indispensable part of a RNA-protein complex carrying out the pre-rRNA processing. Sequence analysis clearly shows that eukaryotic and archaeal enzymes have an extra insert in their C-terminal domain which is absent in bacterial enzymes and a better understanding of Dim1's function is only possible by its structural characterization which is the aim of this study. After several attempts, the yeast Dim1p was expressed under mild conditions in E. coli and purified in soluble form. Dim1p was able to methylate bacterial 30S subunits both in vivo and in vitro, indicating its ability to recognize bacterial substrate. Supporting our hypothesis, neither the bacterial nor archaeal orthologs were able to complement the processing function of Dim1p in yeast, tested using the plasmid shuffling technique. Our results suggest that the C-terminal insert of Dim1p, along with some structural features of the N-terminal domain, is important for its function in pre-rRNA processing. Further studies are required to understand the complex interactions between proteins and RNA involved in the ribosome biogenesis.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1976 |
| Date | 01 January 2008 |
| Creators | Pulicherla, Nagesh |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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