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.
Highlights
Radiochemicals and x-ray films were obtained from Amersham (Braunschweig, Germany), Kirromycin was a gift from A, Parmeggiani (Palaiseau, France), other antibiotics were from Serva (Heidelberg, Germany) and nucleotides from Boehringer (Mannheim, Germany)
Inhibition of the phosphorylation by kirromycin is a direct EF-Tu-correlated effect and not mediated by inhibition of the kinase as can be concluded from experiments in which EF-TuAR was phosphorylated by wild-type S15 (MRE 600) in the presence of kirromycin
The inability of the mutant strain kinase to modify the wild-type EF-Tu underlines the importance of the phosphorylation for proper function
Summary
The costs of publication of this article were defrayed in part by the payment of page charges, This article must be hereby marked "advertisement" in accordance with 18 U,S,C, Section 1734 solely to indicate this fact,. (GDP) has to be replaced by GTP in the elongation factor Ts-mediated guanine-nucleotide exchange reaction, EF-Ts is an essential protein for the growth of Escherichia coli and its expression is tightly regulated (Hwang et al 1989) but only little is known about the molecular mechanism of its interaction with EF-Tu (Hwang et al, 1992), Protein biosynthesis is the target of numerous antibiotics (Nierhaus, 1982), The antibiotic kirromycin binds with high affinity to EF-Tu forcing this protein into a conformation that is unable to maintain its proper function during the translational elongation cycle, The affinity of the EF-Tu,GTP, kirromycin complex toward aa-tRNA is drastically reduced (Abrahams et al 1991) while even the GDP form of EF-Tu promotes binding of aa-tRNA to the ribosomal A-site in presence of the antibiotic (Wolf et al 1974), The inhibitory effect of kirromycin on translation is due to the fact that the release of EF-Tu'GDP from the acceptor site ofthe ribosome is prevented (Wolf et al 1977), Crystallographic studies have shown that EF-Tu consists of three distinct domains: domain 1 (residues 1-200), domain 2 (residues 209-299), and domain 3 (residues 300-393) (Kjeldgaard and Nyborg, 1992; Berchtold et al, 1993; Kjeldgaard et al 1993), Analysis of kirromycin-resistant mutants (Zeef and Bosch, 1993) revealed that the antibiotic may interact at the interface of domains 1 and 3 when EF-Tu is in the GTP bound state (Mesters et al, 1994), It is known of the E, coli EF-Tu that the protein is N terminally acetylated (Arai et al 1980; Jones et al 1980) and that lysine 56 can be mono- or dimethylated in vivo (L'ltalien and Laursen, 1979), Recently we have shown that EF-Tu from E, coli and Thermus thermophilus are post-translationally modified by phosphorylation in vivo (Lippmann et al 1991, 1993), Phosphorylation of the eukaryotic EF-1a (the eukaryotic counterpart ofEF-Tu) was described, but the site of modification was not determined (Venema et al, 1991a, 1991b), We identified the site of phosphorylation in E, coli EF-Tu as threonine 382 (Lippmann et al, 1993), This residue is strictly conserved in all known EF-Tu and EF-1a sequences, The modification can be reversed by the action of a phosphatase, Here we report the influence of the phosphorylation on the interaction of EF-Tu with EF-Ts, kirromycin, and aa-tRNA
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