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

Wei Liu; Darius Kavaliauskas; Jared Schrader; Kiran Poruri; Victoria Birkedal; Emanuel Goldman; Hieronim Jakubowski; Wlodek Mandecki; Olke Uhlenbeck; Charlotte Rohde Knudsen; Yale Goldman; Barry Cooperman

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

Wei Liu, Darius Kavaliauskas, Jared Schrader, Kiran Poruri, Victoria Birkedal, Emanuel Goldman, Hieronim Jakubowski, Wlodek Mandecki, Olke Uhlenbeck, Charlotte Rohde Knudsen, Yale Goldman, Barry Cooperman

Interdisciplinary Nanoscience Center - INANO-Fysik, iNANO-huset

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

5 Citations (Scopus)

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

Original language	English
Journal	A C S Chemical Biology
Volume	9
Issue	10
Pages (from-to)	2421-2431
Number of pages	11
ISSN	1554-8929
Publication status	Published - 2014

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@article{121ef8bcd04848ab9d2249a1859d7882,

title = "Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation",

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",

author = "Wei Liu and Darius Kavaliauskas and Jared Schrader and Kiran Poruri and Victoria Birkedal and Emanuel Goldman and Hieronim Jakubowski and Wlodek Mandecki and Olke Uhlenbeck and Knudsen, {Charlotte Rohde} and Yale Goldman and Barry Cooperman",

year = "2014",

language = "English",

volume = "9",

pages = "2421--2431",

journal = "A C S Chemical Biology",

issn = "1554-8929",

publisher = "American Chemical Society",

number = "10",

}

TY - JOUR

T1 - Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation

AU - Liu, Wei

AU - Kavaliauskas, Darius

AU - Schrader, Jared

AU - Poruri, Kiran

AU - Birkedal, Victoria

AU - Goldman, Emanuel

AU - Jakubowski, Hieronim

AU - Mandecki, Wlodek

AU - Uhlenbeck, Olke

AU - Knudsen, Charlotte Rohde

AU - Goldman, Yale

AU - Cooperman, Barry

PY - 2014

Y1 - 2014

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

M3 - Journal article

SN - 1554-8929

VL - 9

SP - 2421

EP - 2431

JO - A C S Chemical Biology

JF - A C S Chemical Biology

IS - 10

ER -

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

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