Phosphorylation of Elongation Factor Tu Prevents Ternary Complex Formation

Abstract

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.

Eukaryote-Like Serine/Threonine Kinases and Phosphatases in Bacteria

“…A number of in vitro targets of PrpC are proteins involved in translation, including EF-Tu, EF-G, and CpgA (1,50). EF-Tu is also a substrate of the eSTP Stp in Listeria monocytogenes (4,5). Thus, while eSTPs appear to modulate processes involved in growth, the mechanistic basis of this regulation is not well understood.…”

Structural Insights Into Translational Fidelity

“…Although there is no indication that the ribosome directly inserts a catalytic residue, there are also no convincing candidates in EFTu itself, as judged by crystal structures and mutational studies (168,169). It is also known that kirromycin and the closely related aurodox somewhat stimulate the GTPase activity of EF-Tu even in the absence of the ribosome (170). On this basis, the crystal structure of EF-Tu:GDP bound to aurodox suggests that a particular histidine residue (His84) might contribute to transition state stabilization during GTP hydrolysis (138).…”

Translation Elongation Factor 1A Mutants with Altered Actin Bundling Activity Show Reduced Aminoacyl-tRNA Binding and Alter Initiation via eIF2α Phosphorylation

“…Previous studies have shown that eEF1A is phosphorylated in response to different growth conditions (34,35). The negative impact on eEF1A function as a result of phosphorylation may be through defective aa-tRNA binding (36). Phosphoproteomic studies in S. cerevisiae have identified several conserved residues that are phosphorylated (37-39).…”