Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation

Liu, Wei; Kavaliauskas, Darius; Schrader, Jared M.; Poruri, Kiran; Birkedal, Victoria; Goldman, Emanuel; Jakubowski, Hieronim; Mandecki, Włodek; Uhlenbeck, Olke C.; Knudsen, Charlotte R.; Goldman, Yale E.; Cooperman, Barry S.

doi:10.1021/cb500409y

Cited by 7 publications

(22 citation statements)

References 48 publications

(139 reference statements)

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“…70SIC Cy3 and 70SIC Cy5 ( 16 ), EF-Tu QSY9 , EF-Tu Cy3 , EF-Tu Cy5 and EF-Tu AV-Cy5 ( 15 ), Phe-tRNA Phe (Cy3) and Phe-tRNA Phe (Cy5) ( 17 ), labeled TCs ( 15 ) and fMet-tRNA fMet (prf) and coumarin-labeled phosphate binding protein (C-PBP) ( 18 ) were prepared using described procedures.…”

Section: Methodsmentioning

confidence: 99%

“…Labeled initiation complexes, denoted 70SIC Cy3 and 70SIC Cy5 , contained protein L11 labeled at position 87 with either Cy3 or Cy5, respectively. EF-Tu derivatives prepared from the single-mutation variant of EF-Tu, E348C-EF-Tu, are denoted EF-Tu QSY9 (QSY9 is a dark fluorescence quencher), EF-Tu Cy3 and EF-Tu Cy5 ( 15 ). A fourth derivative, denoted EF-Tu AV-Cy5 , was prepared from the triple-mutation variant C137A/C255V/E348C-EF-Tu ( 15 ).…”

Section: Methodsmentioning

confidence: 99%

“…EF-Tu derivatives prepared from the single-mutation variant of EF-Tu, E348C-EF-Tu, are denoted EF-Tu QSY9 (QSY9 is a dark fluorescence quencher), EF-Tu Cy3 and EF-Tu Cy5 ( 15 ). A fourth derivative, denoted EF-Tu AV-Cy5 , was prepared from the triple-mutation variant C137A/C255V/E348C-EF-Tu ( 15 ). tRNAs labeled at dihydrouridine positions in the D-loop are denoted Phe-tRNA Phe (Cy3), Phe-tRNA Phe (Cy5) and fMet-tRNA fMet (prf), where prf is an abbreviation for proflavin.…”

Section: Methodsmentioning

confidence: 99%

“…All complexes were prepared in buffer A (50 mM Tris-HCl [pH7.5], 70 mM NH 4 Cl, 30 mM KCl, 7 mM MgCl 2 and 1 mM DTT) at 37°C as described ( 15 ). 70SICs were programmed with mRNA 022 ( 19 ).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, we described the development of two fluorescence resonance energy transfer (FRET) assays monitoring, during PRE complex formation, the proximity of EF-Tu to the D-loop of aa-tRNA (EF-Tu:tRNA assay) and to the C-terminal domain (residue 87) of ribosomal protein L11 ( 15 ), which lies within the GAC (EF-Tu:L11 assay). These assays utilize functionally active fluorescent and fluorescence quencher derivatives of EF-Tu labeled at position 348 (Supplementary Figure S1).…”

Section: Introductionmentioning

confidence: 99%

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EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

et al. 2015

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The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

et al. 2015

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Visualization of translation and protein biogenesis at the ER membrane

Gemmer

Chaillet

Loenhout

et al. 2023

Nature

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The dynamic ribosome–translocon complex, which resides at the endoplasmic reticulum (ER) membrane, produces a major fraction of the human proteome1,2. It governs the synthesis, translocation, membrane insertion, N-glycosylation, folding and disulfide-bond formation of nascent proteins. Although individual components of this machinery have been studied at high resolution in isolation3–7, insights into their interplay in the native membrane remain limited. Here we use cryo-electron tomography, extensive classification and molecular modelling to capture snapshots of mRNA translation and protein maturation at the ER membrane at molecular resolution. We identify a highly abundant classical pre-translocation intermediate with eukaryotic elongation factor 1a (eEF1a) in an extended conformation, suggesting that eEF1a may remain associated with the ribosome after GTP hydrolysis during proofreading. At the ER membrane, distinct polysomes bind to different ER translocons specialized in the synthesis of proteins with signal peptides or multipass transmembrane proteins with the translocon-associated protein complex (TRAP) present in both. The near-complete atomic model of the most abundant ER translocon variant comprising the protein-conducting channel SEC61, TRAP and the oligosaccharyltransferase complex A (OSTA) reveals specific interactions of TRAP with other translocon components. We observe stoichiometric and sub-stoichiometric cofactors associated with OSTA, which are likely to include protein isomerases. In sum, we visualize ER-bound polysomes with their coordinated downstream machinery.

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Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome

Kavaliauskas

Chen

Liu

et al. 2018

Nucleic Acids Research

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The GTPase elongation factor EF-Tu delivers aminoacyl-tRNAs to the mRNA-programmed ribosome during translation. Cognate codon-anticodon interaction stimulates GTP hydrolysis within EF-Tu. It has been proposed that EF-Tu undergoes a large conformational change subsequent to GTP hydrolysis, which results in the accommodation of aminoacyl-tRNA into the ribosomal A-site. However, this proposal has never been tested directly. Here, we apply single-molecule total internal reflection fluorescence microscopy to study the conformational dynamics of EF-Tu when bound to the ribosome. Our studies show that GTP hydrolysis initiates a partial, comparatively small conformational change of EF-Tu on the ribosome, not directly along the path from the solution ‘GTP’ to the ‘GDP’ structure. The final motion is completed either concomitant with or following dissociation of EF-Tu from the ribosome. The structural transition of EF-Tu on the ribosome is slower when aa-tRNA binds to a cognate versus a near-cognate codon. The resulting longer residence time of EF-Tu on the ribosome may be important for promoting accommodation of the cognate aminoacyl-tRNA into the A-site.

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Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation

Cited by 7 publications

References 48 publications

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

Visualization of translation and protein biogenesis at the ER membrane

Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome

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