EF-Tu undergoes major conformational changes upon GTP hydrolysis. Unlike other GTP-binding proteins, EF-Tu exhibits a dramatic conformational change in the effector region, involving an unwinding of a small helix and the formation of a beta hairpin structure. This change is presumably involved in triggering the release of tRNA, and EF-Tu, from the ribosome.

The elongation factor Tu (EF-Tu) is a member of the GTP/GDP-binding proteins and interacts with various partners during the elongation cycle of protein biosynthesis thereby mediating the correct binding of amino-acylated transfer RNA (aa-tRNA) to the acceptor site (A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its GDP-bound state. In vivo, EF-Tu is post-translationally modified by phosphorylation. Here we report that the phosphorylation of EF-Tu by a ribosome associated kinase activity is drastically enhanced by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function, inhibits the modification. This effect is specific, since kirromycin-resistant mutants do become phosphorylated in the presence of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu does not bind kirromycin. Most interestingly, the phosphorylation of EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation the site of modification is located at the interface between domains 1 and 3 and is involved in a strong interdomain hydrogen bond. Introduction of a charged phosphate group at this position will change the interaction between the domains, leading to an opening of the molecule reminiscent of the GDP conformation. A model for the function of EF-Tu phosphorylation in protein biosynthesis is presented.

EF-Tu from E. coli, one of the superfamily of GTPase switch proteins, plays a central role in the fast and accurate delivery of aminoacyl-tRNAs to the translating ribosome. An overview is given about the regulatory effects of methylation, phosphorylation and phage-induced cleavage of EF-Tu on its function. During exponential growth, EF-Tu becomes monomethylated at Lys56 which is converted to Me2Lys upon entering the stationary phase. Lys56 is in the GTPase switch-1 region (residues 49-62), a strongly conserved site involved in interactions with the nucleotide and the 5' end of tRNA. Methylation was found to attenuate GTP hydrolysis and may thus enhance translational accuracy. In vivo 5-10% of EF-Tu is phosphorylated at Thr382 by a ribosome-associated kinase. In EF-Tu-GTP, Thr382 in domain 3 has a strategic position in the interface with domain 1; it is hydrogen-bonded to Glu117 that takes part in the switch-2 mechanism, and is close to the T-stem binding site of the tRNA, in a region known for many kirromycin-resistance mutations. Phosphorylation is enhanced by EF-Ts, but inhibited by kirromycin. In reverse, phosphorylated EF-Tu has an increased affinity for EF-Ts, does not bind kirromycin and can no longer bind aminoacyi tRNA. The in vivo role of this reversible modification is still a matter of speculation. T4 infection of E. coli may trigger a phase-exclusion mechanism by activation of Lit, a host-encoded proteinase. As a result, EF-Tu is cleaved site-specifically between Gly59-Ile60 in the switch-1 region. Translation was found to drop beyond a minimum level. Interestingly, the identical sequence in the related EF-G appeared to remain fully intact. Although the Lit cleavage-mechanism may eventually lead to programmed cell death, the very efficient prevention of phage multiplication may be caused by a novel mechanism of in cis inhibition of late T4 mRNA translation.

The structure of the RNA duplex r(CUGGGCGG).r(CCGCCUGG) has been determined at 1.6 A resolution and refined to a final R factor of 18.3% (R(free) = 24.1%). The sequence of the RNA fragment resembles domain E of Thermus flavus 5S rRNA. A previously undescribed wobble-like G.C base-pair formation is found. Owing to the observed hydrogen-bond network, it is proposed that the cytosine is protonated at position N3. The unusual base-pair formation is presumably strained by intermolecular interactions. In this context, crystal packing and particular intermolecular contacts may have direct influence on the three-dimensional structure. Furthermore, this structure includes two G.U wobble base pairs in tandem conformation, with the purines forming a so-called 'cross-strand G stack'.

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