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 |
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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. |