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...
During protein synthesis the interaction with ribosomes of elongation factors Tu (EF-Tu), G (EF-G) and initiation factor 2 (IF-2) is associated with the hydrolysis of GTP which is directly related to the functions of the factors. In this article we review systematically the properties of these GTPase activities in the presence and absence of protein synthesis, and by examining the characteristics of the different minimal systems for the expression of these activities we point to the role of the various effectors and to the enzymological aspects of the systems. For EF-Tu, it has been possible to eliminate any requirement for macromolecular effectors, showing that the factor itself is a GTPase. For EF-G, the presence of at least the 50S ribosomal subunit has remained a requirement, whereas IF-2 needs both the 50S and 30S subunits to exhibit GTPase activity. Between the GTPase activities of the three factors there are some striking similarities, but important differences prevail as a consequence of the specificity of the different functions. This can also be seen by examining the respective ribosomal regions implicated in these reactions. When coupled with protein synthesis, the three GTPase activities reveal characteristics differing from those observed in partial systems.
A series of ribosomal subparticles derived from the 50S subunit has been studied and compared in EF-T- and EF-G-dependent reactions. Three different 50S cores were prepared by CsC1 isophycnic centrifugation and one by NH(4)Cl-ethanol extractionm the 50S CsCl core a had lost proteins L1, L7, L8, L10, L12, L16, L25, L33, and some L6 and L11. The 50S CsCl core b additionally lacked protein L6, and 50S CsCl core c also lacked protein L5, L15, L18, L27, L28, L30, and most of L9, L14, L19, and L21. The 50S NH(4)Cl-ethanol core had lost up to 90 percent of proteins L7, L12 and 30-60 percent of proteins L8, L10, and L29. The 50S CsCl core a had much reduced activity in EF-G and none in EF-T GTPase reactions while 50S CsCl cores b and c were inactive. Addition of proteins L7, L12 restored the activity for both the EF-T- and EF-G-dependent GTPase with all of the three 50S CsCl cores, increasing stepwise from core c to core a; The 50S NH(4)Cl-ethanol core was partially active in the EF-G GTPase over the 2-30 mM MG-2+ range tested, while EF-T only showed some activity inthe upper portion of this range...
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