Abstract
During protein synthesis, elongation factor-Tu (EF-Tu) bound to GTP chaperones the entry of aminoacyl-tRNA (aa-tRNA) into actively translating ribosomes. In so doing, EF-Tu increases the rate and fidelity of the translation mechanism. Recent evidence suggests that EF-Ts, the guanosine nucleotide exchange factor for EF-Tu, directly accelerates both the formation and dissociation of the EF-Tu-GTP-Phe-tRNA(Phe) ternary complex (Burnett, B. J., Altman, R. B., Ferrao, R., Alejo, J. L., Kaur, N., Kanji, J., and Blanchard, S. C. (2013) J. Biol. Chem. 288, 13917-13928). A central feature of this model is the existence of a quaternary complex of EF-Tu/Ts·GTP·aa-tRNA(aa). Here, through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species. These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions.
Highlights
Elongation factor-Tu (EF-Tu) chaperones aminoacyl-tRNA to the elongating ribosome as a ternary complex with guanosine triphosphate (GTP)
We measured the apparent affinity of highly purified, nucleotide-free EF-Tu or an EF-Tu/Ts complex for three distinct elongator tRNA species, each containing the same acp3U post-transcriptional modification at position 47 used for Cy3 labeling: Phe-tRNAPhe, Met-tRNAMet, and Arg
After aa-tRNA is delivered to the actively translating ribosome, EF-Tu1⁄7GDP is released as an inactive species whereupon the guanosine nucleotide exchange factor, elongation factor Ts (EF-Ts), operates on it to exchange GDP for GTP
Summary
Elongation factor-Tu (EF-Tu) chaperones aminoacyl-tRNA (aa-tRNA) to the elongating ribosome as a ternary complex with GTP. Through comparative investigations of phenylalanyl, methionyl, and arginyl ternary complexes, and the development of a strategy to monitor their formation and decay using fluorescence resonance energy transfer, we reveal the generality of this newly described EF-Ts function and the first direct evidence of the transient quaternary complex species These findings suggest that EF-Ts may regulate ternary complex abundance in the cell through mechanisms that are distinct from its guanosine nucleotide exchange factor functions. We directly examined whether such findings are general in nature by measuring the impact of EF-Ts on the kinetic properties of distinct ternary complex species that exhibit distinct apparent KD values [29] These investigations, combined with fluorescence resonance energy transfer (FRET) methods that facilitated the direct monitoring of EF-Tu/Ts interactions, yielded a general kinetic framework that explains how EF-Ts may influence the thermodynamic compensation model underpinning ternary complex stability, why quaternary complex has escaped detection previously, and. How cellular interactions between EF-Ts and ternary complex can influence the regulation of protein synthesis in the cell
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