Functional studies of elongation factor Tu from Escherichia coli : Site-directed mutagenesis and antibiotic actio

Abstract

This PhD thesis describes several studies into the structure and function of Escherichia coli Elongation Factor Tu (EF-Tu). EF-Tu plays a central role in the bacterial protein synthesis machinery as the carrier of "coded building blocks" for protein synthesis, aminoacylated tRNA (aa-tRNA). Without EF-Tu, fast and accurate protein synthesis would be impossible. It not only binds and carries the aa-tRNA to the ribosome, it also plays a role in the selection of the correct aa-tRNA for the codon of the messenger RNA (mRNA) currently being decoded. This selection process, which takes place on the ribosome, is supported by hydrolysis of a GTP molecule bound to the EF-Tu-aa-tRNA complex. Once the GTP is hydrolyzed to GDP, the EF-Tu changes conformation and detaches from both the aa-tRNA and the ribosome. The aa-tRNA, if it matches the anticodon on the mRNA, will progress to the incorporation of its activated amino acid into the growing protein chain. If the anticodon does not match, however, proofreading will (r)eject it from the ribosome. There are indications that a second EF-Tu, or at least GTP hydrolysis by a second EF-Tu is involved in this process, but it is not understood how. The work in this thesis is mostly centered on understanding the role of several amino acid residues of EF-Tu in the interaction with the GTP or GDP nucleotide, the catalysis of GTP hydrolysis but EF-Tu and the mechanism by which this only accelerated five orders of magnitude on the ribosome. It leads to a number of interesting conclusions that shed light on the problem, but cannot explain the "triggered catalysis" mechanism.