The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules.
The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D010444:Peptide Elongation Factor Tu; D010445:Peptide Elongation Factors / Q000378:metabolism*; D011485:Protein Binding; D011728:Pyridones / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D046911:Macromolecular Substances; D010445:Peptide Elongation Factors; D011728:Pyridones; D012346:RNA, Transfer, Amino Acyl; D006153:Guanosine Diphosphate; D014108:Tosylphenylalanyl Chloromethyl Ketone; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D010444:Peptide Elongation Factor Tu; D005033:Ethylmaleimide; C008536:mocimycin