In Vivo Deuteration of Transfer RNAs: Overexpression and Large-Scale Purification of Deuterated Specific tRNAs

Jünemann, Ralf; Wadzack, Jörg; Triana-Alonso, Francisco J.; Bittner, J.; Caillet, J.; Meinnel, Thierry; Vanatalu, Kalju; Nierhaus, Knud H.

doi:10.1093/nar/24.5.907

Cited by 41 publications

(22 citation statements)

References 25 publications

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“…The deuteration of recombinant proteins expressed in Escherichia coli and the methylotropic yeast Pichia pastoris has been reported (4,5,8,9,12,14). They can grow in high concentration of D 2 O, and their components are labeled with D in exchangeable sites.…”

Section: Discussionmentioning

confidence: 99%

Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D₂O Medium

Unno

Hagima

Kishido

et al. 2005

Appl Environ Microbiol

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Fully deuterated components from autotrophic cell lysate are useful materials for labeling of heterotrophs with deuterium. To facilitate the faster production of deuterated algal lysate, we selected a mutant Chlorella strain that grows faster in heavy water than the wild type. The mutant DR-17 was found to have a higher level of Hsp60 and an elevated level of protein synthesis. We previously isolated a deuterium-resistant yeast cell line that was also found to express elevated level of Hsp70 (K. Unno, T. Kishido, M. Morioka, S. Okada, and N. Oku, Biol. Pharm. Bull. 26:799-802, 2003). This suggests that the overexpression of heat shock proteins is required to compensate for the deuterium isotope effect.Stable isotope labeling is an essential tool for studying the functional structure and assembly of proteins by nuclear magnetic resonance (NMR). While high deuteration levels of nonexchangeable sites in a protein are beneficial for main chain assignment, the complete exchange of D in exchangeable sites to H is required for proton detection. If amide protons involved in strong hydrogen bonds or buried inside the protein are not accessible to H in the solvent, the amides will remain in the deuterated form. Although this problem may be resolved by unfolding the protein using chemical denaturants followed by refolding in the presence of H 2 O, complete exchange and refolding is not always possible.The expression of proteins in fully deuterated algal lysate medium in 100% H 2 O has recently been described (6). Using this technique, deuterated samples were uniformly protonated at their amide sites, irrespective of the solvent exchange characteristics of the folded protein. We isolated a mutant clone to facilitate the production of deuterated growth media from algal Chlorella. 14 C-␣ aminoisobutyric acid (␣-AIB), which is an unavailable amino acid for protein synthesis, in 60% D 2 O medium at 25°C. The relative radioactivity of each precursor was 18.5 kBq/0.2 M/ml. The incubated cells were washed with icecold water. The incorporation of amino acid into cells was determined from the radioactivity of cells using a liquid scintillation counter (LSC). That of nucleic acids was determined from the radioactivity in the ethanol-insoluble fraction of cells. MATERIALS AND METHODS PreparationThe effect of D 2 O on protein synthesis was investigated using 35 S-L-methionine ( 35 S-Met; 185 kBq/0.2 M/ml) in 60 or 100% D 2 O medium. Labeled cells (2 ϫ 10 8 cells) were homogenized with glass beads. The homogenate was centrifuged at 200 ϫ g for 10 min and then at 9,000 ϫ g for 30 min, and the supernatant was applied to a Sephadex G-25 column (PD-10; Pharmacia Biotech). The radioactivity and protein concentration of these fractions were measured using LSC and a protein assay kit (Bio-Rad), respectively.Carbon fixation and amount of photosynthetic pigments. Fixation of 14 C was measured as described previously (13). Briefly, Chlorella cells (2 ϫ 10 7 cells/190 l) were preilluminated with 2.5 klx for 3 min at 25°C, and then 10 l of 10 mM Na...

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Section: Discussionmentioning

confidence: 99%

Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D₂O Medium

Unno

Hagima

Kishido

et al. 2005

Appl Environ Microbiol

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“…The start codon in the P site is bold, and the A-site codon is underlined. The fMet-tRNA fMet and the Phe-tRNA Phe were prepared as previously described (39). We used the same L9-EF-4 protein fusion, expression, and purification procedures as described previously (14).…”

Section: Methodsmentioning

confidence: 99%

Elongation factor 4 remodels the A-site tRNA on the ribosome

Gagnon

Lin

Steitz

2016

Proc. Natl. Acad. Sci. U.S.A.

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During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5′-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.T ranslation of the genetic information requires protein factors that interact with the ribosome sequentially, regulate its activity, and guide it through the protein synthesis cycle in a concerted manner. Many of those factors are guanosine triphosphatases (GTPases), proteins that use energy from guanosine 5′-triphosphate (GTP) to promote conformational changes that lead to transitions between ribosome functional states (1, 2). In bacteria, for instance, initiation of protein synthesis is largely regulated by initiation factor 2 (IF-2), a GTPase that stabilizes the initiator tRNA in the P site of the ribosome (3). Subsequently, the elongation step is catalyzed by two universally conserved GTPases, elongation factor Tu (EF-Tu) and elongation factor G (EF-G). The ternary complex, consisting of EF-Tu, GTP, and the aminoacyl-tRNA, interacts with the ribosome to decode the codon in the A site of the ribosome. Following accommodation of the aminoacyl-tRNA in the A site of the ribosome and subsequent peptide bond formation, the tRNA-mRNA duplex is translocated by one codon-a process catalyzed by EF-G and GTP (4-6). Termination of protein synthesis is triggered when a stop codon is reached, upon which the newly synthesized protein is released with the help of release factor 3 (RF-3), yet another GTPase (7).Elongation factor 4 (EF-4/LepA) is a highly conserved protein structurally similar to EF-G (8) and has a ribosome-dependent GTPase activity (9-13). However, despite numerous studies, its function has remained elusive (9-20). Fast kinetic studies showed that EF-4 competes with EF-G during elongation for binding to the pretranslocation (PRE) ribosome, with tRNAs in the A and P sites (17). Despite this, EF-4 was also shown to increase the rate of protein synthesis at high intracellular ionic strength (16), without any effect on translational accuracy (16, 18). Conversely, EF-4 was also reported...

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“…Tth 70S ribosomes, unmodified tRNA i Met and tRNA Phe from E. coli were purified as previously described (Junemann et al, 1996; Polikanov et al, 2012; Schmitt et al, 1999). Synthetic mRNA with the sequence 5’-GGC AAG GAG GUA AAA AUG UUC UAA-3’ was obtained from IDT (Coralville, IA).…”

Section: Methodsmentioning

confidence: 99%

Amicoumacin A Inhibits Translation by Stabilizing mRNA Interaction with the Ribosome

et al. 2014

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SUMMARY We demonstrate that the antibiotic amicoumacin A (AMI) whose cellular target was unknown, is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G.

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In Vivo Deuteration of Transfer RNAs: Overexpression and Large-Scale Purification of Deuterated Specific tRNAs

Cited by 41 publications

References 25 publications

Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D₂O Medium

Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D₂O Medium

Elongation factor 4 remodels the A-site tRNA on the ribosome

Amicoumacin A Inhibits Translation by Stabilizing mRNA Interaction with the Ribosome

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In Vivo Deuteration of Transfer RNAs: Overexpression and Large-Scale Purification of Deuterated Specific tRNAs

Cited by 41 publications

References 25 publications

Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D2O Medium

Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D2O Medium

Elongation factor 4 remodels the A-site tRNA on the ribosome

Amicoumacin A Inhibits Translation by Stabilizing mRNA Interaction with the Ribosome

Contact Info

Product

Resources

About

Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D₂O Medium

Deuterium-Resistant Algal Cell Line for D Labeling of Heterotrophs Expresses Enhanced Level of Hsp60 in D₂O Medium