TY - JOUR
T1 - Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation
AU - Liu, Wei
AU - Kavaliauskas, Darius
AU - Schrader, Jared M.
AU - Poruri, Kiran
AU - Birkedal, Victoria
AU - Goldman, Emanuel
AU - Jakubowski, Hieronim
AU - Mandecki, Wlodek
AU - Uhlenbeck, Olke C.
AU - Knudsen, Charlotte R.
AU - Goldman, Yale E.
AU - Cooperman, Barry S.
N1 - 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.
PY - 2014/12/31
Y1 - 2014/12/31
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84908214110&partnerID=8YFLogxK
UR - https://pubs.acs.org/doi/abs/10.1021/cb500409y
U2 - 10.1021/cb500409y
DO - 10.1021/cb500409y
M3 - Article
AN - SCOPUS:84908214110
SN - 1554-8929
VL - 9
SP - 2421
EP - 2431
JO - ACS Chemical Biology
JF - ACS Chemical Biology
IS - 10
ER -