GE2270A-resistant mutations in elongation factor Tu allow productive aminoacyl-tRNA binding to EF-Tu.GTP.GE2270A complexes.

Abstract

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.

• Authors: A M Zuurmond, J Martien de Graaf, L N Olsthoorn-Tieleman, B Y van Duyl, V G Mörhle, F Jurnak, J R Mesters, R Hilgenfeld, B Kraal
• Journal: Journal of molecular biology (J Mol Biol) Vol. 304 Issue 5 Pg. 995-1005 (Dec 15 2000) ISSN: 0022-2836 [Print] Netherlands
• PMID: 11124042 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright 2000 Academic Press.

Chemical References

• Aminoglycosides
• Anti-Bacterial Agents
• Peptides
• Peptides, Cyclic
• RNA, Bacterial
• RNA, Transfer, Amino Acyl
• Thiazoles
• pulvomycin
• Guanosine Diphosphate
• Poly U
• polyphenylalanine
• Guanosine Triphosphate
• Peptide Elongation Factor Tu
• Adenine
• GE 2270 A

Topics

• Actinomycetales (chemistry)
• Adenine (metabolism)
• Amino Acid Substitution (genetics)
• Aminoglycosides
• Anti-Bacterial Agents (chemistry, pharmacology)
• Drug Resistance, Microbial
• Escherichia coli (chemistry, drug effects, genetics)
• Guanosine Diphosphate (metabolism)
• Guanosine Triphosphate (metabolism)
• Models, Molecular
• Mutation (genetics)
• Peptide Elongation Factor Tu (chemistry, genetics, metabolism)
• Peptides (metabolism)
• Peptides, Cyclic (chemistry, pharmacology)
• Poly U (genetics, metabolism)
• Protein Binding (drug effects)
• Protein Biosynthesis (drug effects)
• Protein Conformation
• RNA, Bacterial (genetics, metabolism)
• RNA, Transfer, Amino Acyl (genetics, metabolism)
• Thermodynamics
• Thermus (chemistry)
• Thiazoles (chemistry, metabolism, pharmacology)