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

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.

Publication DOI: https://doi.org/10.1021/cb500409y
Divisions: Life & Health Sciences > Biosciences
Additional Information: 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.
Uncontrolled Keywords: Biochemistry,Molecular Medicine
Full Text Link:
Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
https://pubs.ac ... .1021/cb500409y (Publisher URL)
Published Date: 2014-12-31
Authors: Liu, Wei
Kavaliauskas, Darius
Schrader, Jared M.
Poruri, Kiran
Birkedal, Victoria
Goldman, Emanuel
Jakubowski, Hieronim
Mandecki, Wlodek
Uhlenbeck, Olke C.
Knudsen, Charlotte R.
Goldman, Yale E.
Cooperman, Barry S.

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