Modification of Elongation‐Factor‐Tu · Guanine‐Nucleotide Interaction by Kirromycin

Abstract

Kirromycin activates functions of elongation factor Tu (EF-Tu) that normally require the presence of specific effectors [Wolf et al. (1974) Proc. Nut1 Acud. Sci. U.S.A. 71, 4910-49141. As a result, the EF-Tu GTPase activity is uncoupled from aminoacyl-tRNA and ribosomes. For a better understanding of the action of the antibiotic, we have studied its effect on the interaction between EF-Tu and guanine nucleotides and compared this with the action of the physiological effectors aminoacyltRNA and elongation factor Ts (EF-Ts). Kirromycin affects both association (k; and dissociation (k' 1) rates of EF-Tu and GTP, but in opposite ways, the k: being strongly increased, whereas the kL is even more strongly decreased. This causes a lowering of the apparent K ' of this complex by two orders of magnitude, i.e. approaching that of EF-Tu . GDP. By contrast, the k : 1 and k l 1 are both increased to the same extent and consequently the apparent K ' of this complex is unchanged in the presence of the antibiotic. Thus the action of kirromycin resembles that of EF-Ts opening the EF-Tu site for GDP, thereby increasing the rate of the EF-Tu.GDP/GDP exchange, but in contrast to EF-Ts kirromycin binds preferentially to EF-Tu . GTP. Like aminoacyl-tRNA, the antibiotic induces a specific conformation that locks GTP in its site on EF-Tu. The regeneration of EF-Tu . GTP from EF-Tu .GDP does not limit the rate of GTP hydrolysis induced by kirromycin, even when aminoacyl-t RNA or ribosomes' stimulate this reaction. By contrast, the turnover activity occurring in the absence of the antibiotic and depending on aminoacyl-tRNA plus ribosomes is limited by the dissociation rate of the EF-Tu.GDP complex. Our results indicate that kirromycin induces a conformation of EF-Tu that shares features with the conformations evoked sequentially by EF-Ts, aminoacyl-tRNA and ribosomes during the elongation cycle.In each round of polypeptide chain elongation, the elongation factor Tu (EF-Tu) forms a ternary complex with GTP and aminoacyl-tRNA (aa-tRNA) that interacts with mRNA .ribosomes, resulting in the binding of aa-tRNA to the ribosome, hydrolysis of GTP and release of EF-Tu.GDP (for a review, see [ I -31). The regeneration of the ternary complex from EF-Tu .GDP is accelerated by elongation factor Ts (EF-Ts). We have shown that kirromycin, an inhibitor of protein biosynthesis and the first antibiotic having EF-Tu as its target, binds to EF-Tu in a one-to-one ratio and that the EF-Tu . kirromycin complex thus formed can still react sequentially with GTP and aa-tRNA leading to the formation of a quaternary complex [4-81. However, after the enzymatic binding of this complex to the ribosome.mRNA and associated GTP hydrolysis, the modifications introduced by kirromycin in the interactions between EF-Tu, guanine nucleotides and aa-tRNA inhibit the dissociAbbreviations. EF-Tu, elongation factor Tu; EF-Ts, elongation factor Ts; k;, , apparent association rate constant; kl, , apparent dissociation rate constant; aa-tRNA, aminoacyl transfer RNA. ation of EF-Tu f...

Allosteric changes of the guanine nucleotide site of elongation factor EF‐Tu

“…Nucleotide-free EF-Tu was prepared as in [12]. The specific activities of [7-32p]GTP and [3H]GDP were determined by isotopic dilution.…”

“…Dissociation rate constants, association rate constants and GTPase activity were determined as in [12]. Details are in the legends to tables and figures.…”

“…The most important difference found with regard to the mutant EF-TUD2216 was an intrinsically increased affinity for GTP. This mutant protein also displays considerable GTPase activity in the absence of any macromolecular effector, whereas the wild-type does so only in the presence of the antibiotic [12]. It was later demonstrated that the endogenous GTPase activity of the mutant was sustained by the increased affinity for GTP [14].…”

“…This antibiotic inhibits the elongation cycle of protein biosynthesis by preventing the release of EF-Tu from the ribosome after hydrolysis of GTP, thus blocking the ribosome on the messenger RNA [9][10][11]. Kirromycin strongly increases the binding affinity of EF-Tu for GTP [12] and gives rise to a turnover GTPase activity of the protein [9].…”

“…The EF-Tu-guanine nucleotide interaction of various wild-type EF-Tu's and mutant EF-TuD2216 in the presence of several ligands such as kirromycin, the Abbreviation: EF-Tu, elongation factor EF-Tu; subscripts refer to the strain from which the protein was isolated Address correspondence to A. P. elongation factor EF-Ts, Phe-tRNA l'he and/or ribosomes, and under the influence of mono-and divalent cations has been studied [12][13][14]17]: the GTPase centre of the molecule is activated in the presence of increasing monovalent cation concentrations; this activation is stimulated by NI-I~ up to a concentration of 800 mM, where it reaches a plateau [14]. The most important difference found with regard to the mutant EF-TUD2216 was an intrinsically increased affinity for GTP.…”