GTPases are found in all domains of life and are highly conserved. In eukaryotes, they serve as signalling molecules for many cellular processes. However, the prokaryotic GTPases play a very different role and are found to be associated with ribosome function. Among the 11 conserved GTPases, Der is the most interesting in prokaryotes. It possesses a unique structure with two GTPase domains (G-Domains) tethered by a variable length acidic linker and a carboxyl terminal KH-like domain. The exact function of Der is still under investigation and most of the data suggest that it is important for 50S ribosomal assembly or stability. In order to investigate the function of Escherichia coli Der (Ec-Der), expression plasmids for wild-type and mutated proteins were created and the proteins were successfully expressed. The expression of the mutant protein that lacked G-Domain 1 was toxic to the cells and it was found that some large ribosomal proteins were missing from the ribosomes of these cells. In addition, other macromolecular complexes such as the GroEL/GroES chaperonin appeared not to be assembled under these conditions. The activities of both wild-type and mutated proteins were also tested and found to be dependent on potassium ions (K+), which enhanced nucleotide binding. Additionally, intra-molecular control over nucleotide binding and release was also observed for Ec-Der. The in vitro selection of RNA aptamers with nanomolar affinity for Ec-Der produced aptamers that contained short variable sequences. These aptamers affected the growth of the E. coli cells and caused a change in cellular morphology that had been noted previously during Ec-Der over-expression. Ec-Der showed high affinity (nM) to both selected RNA and the unselected RNA library. The activity of Ec-Der and Era was inhibited in the presence of any sequence of RNA that has the length of greater than 16 nucleotides. RNA was also cross-linked to Ec-Der in the presence of GTP, but not GDP, suggesting that RNA was a regulator of the Ec-Der GTPase cycle. Based on these results, it is speculated that Ec-Der might be involved in more than one function. It may be acting at the level of the membrane (based on cellular morphology reported here and by Hwang and Inouye 2001) and may also take part in processes related to ribosome function. Regulation of protein activity by RNA length has not been predicted or described and this may represent a novel mean of regulation of the Era subfamily of GTPases.
| Identifer | oai:union.ndltd.org:ADTP/205232 |
| Date | January 2008 |
| Creators | Aung-Htut, May Thandar, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Publisher:University of New South Wales. Biotechnology & Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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