Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation

Wei Liu, Darius Kavaliauskas, Jared M. Schrader, Kiran Poruri, Victoria Birkedal, Emanuel Goldman, Hieronim Jakubowski, Wlodek Mandecki, Olke C. Uhlenbeck, Charlotte R. Knudsen, Yale E. Goldman, Barry S. Cooperman*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

Original languageEnglish
Pages (from-to)2421-2431
Number of pages11
JournalACS Chemical Biology
Volume9
Issue number10
Early online date15 Aug 2014
DOIs
Publication statusPublished - 31 Dec 2014

Bibliographical note

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

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    Liu, W., Kavaliauskas, D., Schrader, J. M., Poruri, K., Birkedal, V., Goldman, E., Jakubowski, H., Mandecki, W., Uhlenbeck, O. C., Knudsen, C. R., Goldman, Y. E., & Cooperman, B. S. (2014). Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation. ACS Chemical Biology, 9(10), 2421-2431. https://doi.org/10.1021/cb500409y