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Structural studies of EF-Tu complexes

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

  • Jesper Sanderhoff Johansen, Danmark
  • Molekylærbiologisk Institut
Protein synthesis is a process vital to all living organisms. Protein synthesis occurs on large ribonucleoprotein complexes called ribosomes. Amino acids (aa) are brought to the ribosome as part of a ternary complex consisting of elongation factor EF-Tu, GTP and aminoacyl-tRNA (aa-tRNA). The GTP molecule bound to EF-Tu is hydrolysed during the positioning of the aa-tRNA on the ribosome. Subsequently, the ternary complex dissociates and EF-Tu leaves the aa-tRNA and the ribosome in a GDP bound conformation. Peptide bond formation then occurs on the ribosome with the aa-tRNA as substrate.
A second elongation factor, EF-Ts, catalyse the dissociation of GDP from EF-Tu which in turn allows EF-Tu to bind another GTP molecule. The resulting EF-Tu:GTP complex is then cable of forming a ternary complex with a new aa-tRNA which can then associate with the ribosome.
During my time as a Ph. D. student I have studied the structures of different EF-Tu complexes from E. coli, G. anatolicus and bovine mitochondria primarily by X-ray crystallography.
E. coli EF-Tu has been purified and crystallised in complex with the non-hydrolysable GTP analogue GDPNP. The structure of EF-Tu:GDPNP was determined by X-ray crystallography to a resolution of 2.5 Å. Surprisingly, the overall conformation of E. coli EF-Tu:GDPNP is, unlike previously determined structures of EF-Tu GDP/GDPNP complexes, similar to the GDP bound form rather than the GTP bound form. The results obtained for this project are presented in chapter 2 and in manuscript I.
G. anatolicus is a hyperthermophilic bacteria isolated from a thermal vent. The structure of G. anatolicus EF-Ts has been determined in complex with E. coli EF-Tu by X-ray crystallography to a resolution of 2.8 Å. The structure of G. anatolicus EF-Ts is more similar to the previously determined structures of E. coli and bovine mitochondrial EF-Ts rather than EF-Ts from the thermophilic bacteria T. thermophilus. The structure of G. anatolicus EF-Ts in complex with E. coli EF-Tu is presented in chapter 3 and in manuscript II.
Mammalian mitochondria contain an independent genome which encodes 13 proteins and 22 tRNAs and 2 rRNAs. The mammalian mitochondria have their own specialised translational system for maintaining the synthesis of the 13 proteins. The mammalian mitochondrial protein synthesis resembles the prokaryotic system more than the cytosolic system from eukaryotes. Some of the tRNAs encoded by the mammalian mitochondrial genome possesses a number of unusual features distinguishing them from tRNAs from other systems. Secondary structure a prediction shows that in particular tRNASerGCU has an unusual structure. This tRNA completely lacks the D-loop and has an extended T-loop and anticodon stem when compared to other tRNAs.
This project was initiated in order to study the structure of this unusual tRNA, either in its isolated form or preferentially in complex with mammalian mitochondrial EF-Tu. Bovine mitochondrial EF-Tu was produced by recombinant protein expression in E. coli and tRNASerGCU was produced by in vitro transcription. The ternary complex composed of EF-Tu:GDPNP:tRNASerGCU was reconstituted for crystallisation experiments. Unfortunately, we have not succeeding in crystallising neither the free components nor the ternary complex so far. The results of this project are presented in chapter 3.

OriginalsprogEngelsk
Antal sider182
StatusUdgivet - 1 maj 2011

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