Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA.
We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.
Abrahams
J P
JP
Department of Biochemistry, Leiden University, The Netherlands.
van Raaij
M J
MJ
Ott
G
G
Kraal
B
B
Bosch
L
L
D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000187:drug effects / Q000378:metabolism*; D011728:Pyridones / Q000494:pharmacology; D012346:RNA, Transfer, Amino Acyl / Q000187:drug effects / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D011728:Pyridones; D012346:RNA, Transfer, Amino Acyl; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu; C008536:mocimycin