Abstract:
: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)
journal_name
Biochemistryjournal_title
Biochemistryauthors
Dell VA,Miller DL,Johnson AEdoi
10.1021/bi00459a014subject
Has Abstractpub_date
1990-02-20 00:00:00pages
1757-63issue
7eissn
0006-2960issn
1520-4995journal_volume
29pub_type
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