Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation.
ACS Chem Biol
; 9(10): 2421-31, 2014 Oct 17.
Article
em En
| MEDLINE
| ID: mdl-25126896
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteínas Ribossômicas
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Ribossomos
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Biossíntese de Proteínas
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Aminoacil-RNA de Transferência
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Fator Tu de Elongação de Peptídeos
Tipo de estudo:
Prognostic_studies
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article