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

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

  • Wei Liu, University of Pennsylvania, Philadelphia, PA, United States
  • Darius Kavaliauskas, Denmark
  • Jared Schrader, Northwestern University, Evanston, USA, United States
  • Kiran Poruri, Rutgers, the State University of New Jersey, United States
  • Victoria Birkedal
  • Emanuel Goldman, Rutgers, the State University of New Jersey, United States
  • Hieronim Jakubowski, Rutgers, the State University of New Jersey, United States
  • Wlodek Mandecki, Rutgers, the State University of New Jersey, United States
  • Olke Uhlenbeck, Northwestern University, Evanston, USA, United States
  • Charlotte Rohde Knudsen
  • Yale Goldman, University of Pennsylvania School of Medicine, United States
  • Barry Cooperman, University of Pennsylvania, Philadelphia, PA, United States
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
JournalA C S Chemical Biology
Volume9
Issue10
Pages (from-to)2421-2431
Number of pages11
ISSN1554-8929
Publication statusPublished - 2014

See relations at Aarhus University Citationformats

ID: 84323378