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

Kirromycin activates functions of elongation factor Tu (EF‐Tu) that normally require the presence of specific effectors [Wolf et al. (1974) Proc. Natl Acad. Sci. U.S.A. 71 , 4910–4914]. 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 aminoacyl‐tRNA and elongation factor Ts (EF‐Ts). Kirromycin affects both association ( k′ +1 ) and dissociation ( k′ −1 ) rates of EF‐Tu and GTP, but in opposite ways, the k′ +1 being strongly increased, whereas the k′ ‐1 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′ ‐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‐tRNA 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.