Coupling of GTP hydrolysis by EF-G to tRNA and mRNA translocation through the ribosome

by Carlos Eduardo da Cunha
Doctoral thesis
Date of Examination:2013-06-19
Date of issue:2013-09-20
Advisor:Prof. Dr. Marina Rodnina
Referee:Prof. Dr. Kai Tittmann
Referee:Prof. Dr. Holger Stark
Persistent Address: http://dx.doi.org/10.53846/goediss-4053

 

 

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Abstract

English

Translocation is the concerted movement of the messenger RNA (mRNA) and the transfer RNA (tRNA) through the ribosome. Although spontaneous in vitro, this process is catalyzed by the translational GTPase elongation factor G (EF-G). How EF-G couples the energy released by GTP hydrolysis to translocation is yet unknown. Furthermore, a mechanistic understanding of translocation is also missing, e.g. the timing of tRNA translocation of the two ribosomal subunits is not known. To address these questions, we designed an EF-G mutant that is unable to hydrolyze GTP, EF-G(H91A), while binding to nucleotides and the ribosome with similar affinities as the wild-type. We find that the rate of translocation is reduced in the presence of EF-G(H91A) as compared to the wild-type EF-G, but is higher than spontaneous translocation. H91A also remains strongly bound to the ribosome and does not dissociate even under non-equilibrium conditions. This suggests that EF-G works in a dual energy regime on the ribosome: (i) as a probabilistic molecular motor biasing the forward tRNA movement through conformational constraints, (ii) as switch-type GTPase dissociating from the ribosome once the GDP-bound conformational state is reached. Additionally, using EF-G(H91A), we demonstrate that translocation occurs in a synchronous way on the two ribosomal subunits and that EF-G binding only causes partial 50S subunit translocation. The energy of GTP hydrolysis is coupled to translocation of the 30S subunit and the completion of translocation on the 50S subunit. Together these results show that EF-G plays a dual role during translocation and that GTP hydrolysis has a key role in synchronizing this process.
Keywords: ribosome; elongation factor G; EF-G; GTPase; translocation; ribosome dynamics; ribosome biophysics; synchronicity
 
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