Effects of nucleotide- and aurodox-induced changes in elongation factor Tu conformation upon its interactions with aminoacyl transfer RNA. A fluorescence study.
The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNA(Phe)-Fl8, a functionally active analogue of Phe-tRNA(Phe) with a fluorescein dye convalently attached to the s4U-8 base. The association of EF-Tu.GDP with Phe-tRNA(Phe)-Fl8 resulted in an average increase of 33% in fluorescein emission intensity. This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu.GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu.GDP-containing ternary complex. The Kd for the Phe-tRNA(Phe)-Fl8.EF-Tu.GDP complex was found to average 28.5 microM, more than 33,000-fold greater than the Kd of the Phe-tRNA(Phe)-Fl8.EF-Tu.GTP complex under the same conditions. In terms of free energy, the delta G degree for ternary complex formation at 6 degrees C was -11.5 kcal/mol with GTP and -5.8 kcal/mol with GDP. Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu.GDP from the aa-tRNA on the ribosome. Aurodox (200 microM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 microM, and increased the Kd of the GTP complex by at least 6-fold. The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu.GDP affinity for aa-tRNA and also weakens EF-Tu.GTP affinity for aa-tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Dell
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VA
Department of Chemistry and Biochemistry, University of Oklahoma, Norman 73019.
Miller
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DL
Johnson
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AE
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000899:Aurodox / Q000494:pharmacology*; D004926:Escherichia coli / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007700:Kinetics; D010444:Peptide Elongation Factor Tu / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000900:Anti-Bacterial Agents; D006150:Guanine Nucleotides; D012346:RNA, Transfer, Amino Acyl; D012360:RNA, Transfer, Phe; D000899:Aurodox; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu