Two partners of the ribosome, EF-Tu and LepA

Abstract

The translational GTPases elongation factor Tu (EF-Tu) and LepA modulate the dynamics of tRNA on the ribosome. EF-Tu facilitates the delivery of aminoacyl-tRNA (aa-tRNA) to the translating ribosome and LepA catalyzes the retro-translocation of tRNA•mRNA from the E- and P-sites of the ribosome back to the P- and A-sites. Although an increasing body of structural and biochemical information is available, little is known about the functional cycle of LepA during retro-translocation, the kinetics of EF-Tu dissociation from the ribosome and the rate of EF-Tu conformational change during aa-tRNA delivery. This thesis reports the successful construction and biochemical characterisation of a mutant form of EF-Tu from Escherichia coli ideal for the specific incorporation of fluorescent labels, enabling measurements pivotal for uncovering the rate of EF-Tu conformational change and dissociation from the ribosome. Furthermore, to determine structural components critical for LepA’s function, mutant versions of the protein were constructed and biochemically characterised.