Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation. Journal Article uri icon

Overview

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 date

  • October 1, 2014

has subject area

Date in CU Experts

  • March 13, 2015 12:56 PM

Full Author List

  • Liu W; Kavaliauskas D; Schrader JM; Poruri K; Birkedal V; Goldman E; Jakubowski H; Mandecki W; Uhlenbeck OC; Knudsen CR

author count

  • 12

citation count

  • 0

Other Profiles

International Standard Serial Number (ISSN)

  • 1554-8929

Electronic International Standard Serial Number (EISSN)

  • 1554-8937

Additional Document Info

start page

  • 2421

end page

  • 2431

volume

  • 9

issue

  • 10