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https://www.readbyqxmd.com/read/29447106/challenges-of-site-specific-selenocysteine-incorporation-into-proteins-by-escherichia-coli
#1
Xian Fu, Dieter Söll, Anastasia Sevostyanova
Selenocysteine (Sec), a rare genetically encoded amino acid with unusual chemical properties, is of great interest for protein engineering. Sec is synthesized on its cognate tRNA (tRNA Sec ) by the concerted action of several enzymes. While all other aminoacyl-tRNAs are delivered to the ribosome by the elongation factor Tu (EF-Tu), Sec-tRNA Sec requires a dedicated factor, SelB. Incorporation of Sec into protein requires recoding of the stop codon UGA aided by a specific mRNA structure, the SECIS element. This unusual biogenesis restricts the use of Sec in recombinant proteins, limiting our ability to study the properties of selenoproteins...
February 15, 2018: RNA Biology
https://www.readbyqxmd.com/read/29444814/plastid-translation-elongation-factor-tu-is-prone-to-heat-induced-aggregation-despite-its-critical-role-in-plant-heat-tolerance
#2
Xifeng Li, Chong Cai, Zhe Wang, Baofang Fan, Cheng Zhu, Zhixiang Chen
Translation elongation factor Tu (EF-Tu) is a conserved GTP-binding protein essential for protein translation in prokaryotes and eukaryotic organelles of mitochondria and plastids. EF-Tu also has a GTP/GDP-independent chaperone activity implicated in heat adaptation. Here, we report that Arabidopsis plastid EF-Tu, Rabe1b, rapidly became insoluble in vitro at a temperature as low as 35{degree sign}, but remained largely soluble in vivo at the elevated temperature. At a temperature above 41{degree sign}C, insolubilization of Rabe1b also occurred in vivo, with more than 90% becoming aggregated in 9 hours at 45{degree sign}C...
February 14, 2018: Plant Physiology
https://www.readbyqxmd.com/read/29439212/oxidation-of-translation-factor-ef-tu-inhibits-the-repair-of-photosystem-ii
#3
Haruhiko Jimbo, Rayakorn Yutthanasirikul, Takanori Nagano, Toru Hisabori, Yukako Hihara, Yoshitaka Nishiyama
The repair of photosystem II (PSII) is particularly sensitive to oxidative stress and the inhibition of repair is associated with oxidative damage to the translational elongation system in the cyanobacterium Synechocystis sp. PCC 6803. However, the molecular mechanism of the inhibition of translational elongation remains to be clarified. In a previous study in vitro, we found that EF-Tu, a translation factor that delivers aminoacyl-tRNA to the ribosome, is inactivated by reactive oxygen species via oxidation of Cys82, a single cysteine residue...
February 8, 2018: Plant Physiology
https://www.readbyqxmd.com/read/29366563/pneumococcal-dna-binding-proteins-released-through-autolysis-induce-the-production-of-proinflammatory-cytokines-via-toll-like-receptor-4
#4
Kosuke Nagai, Hisanori Domon, Tomoki Maekawa, Masataka Oda, Takumi Hiyoshi, Hikaru Tamura, Daisuke Yonezawa, Yoshiaki Arai, Mai Yokoji, Koichi Tabeta, Rie Habuka, Akihiko Saitoh, Masaya Yamaguchi, Shigetada Kawabata, Yutaka Terao
Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis...
January 16, 2018: Cellular Immunology
https://www.readbyqxmd.com/read/29339430/simultaneous-binding-of-multiple-ef-tu-copies-to-translating-ribosomes-in-live-escherichia-coli
#5
Mainak Mustafi, James C Weisshaar
In bacteria, elongation factor Tu is a translational cofactor that forms ternary complexes with aminoacyl-tRNA (aa-tRNA) and GTP. Binding of a ternary complex to one of four flexible L7/L12 units on the ribosome tethers a charged tRNA in close proximity to the ribosomal A site. Two sequential tests for a match between the aa-tRNA anticodon and the current mRNA codon then follow. Because one elongation cycle can occur in as little as 50 ms and the vast majority of aa-tRNA copies are not cognate with the current mRNA codon, this testing must occur rapidly...
January 16, 2018: MBio
https://www.readbyqxmd.com/read/29284022/a-transcriptomics-approach-uncovers-novel-roles-for-poly-adp-ribosyl-ation-in-the-basal-defense-response-in-arabidopsis-thaliana
#6
Amy G Briggs, Lori C Adams-Phillips, Brian D Keppler, Sophia G Zebell, Kyle C Arend, April A Apfelbaum, Joshua A Smith, Andrew F Bent
Pharmacological inhibition of poly(ADP-ribose) polymerase (PARP) or loss of Arabidopsis thaliana PARG1 (poly(ADP-ribose) glycohydrolase) disrupt a subset of plant defenses. In the present study we examined the impact of altered poly(ADP-ribosyl)ation on early gene expression induced by the microbe-associate molecular patterns (MAMPs) flagellin (flg22) and EF-Tu (elf18). Stringent statistical analyses and filtering identified 178 genes having MAMP-induced mRNA abundance patterns that were altered by either PARP inhibitor 3-aminobenzamide (3AB) or PARG1 knockout...
2017: PloS One
https://www.readbyqxmd.com/read/29276038/structural-and-dynamics-comparison-of-thermostability-in-ancient-modern-and-consensus-elongation-factor-tus
#7
C Denise Okafor, Manish C Pathak, Crystal E Fagan, Nicholas C Bauer, Megan F Cole, Eric A Gaucher, Eric A Ortlund
Rationally engineering thermostability in proteins would create enzymes and receptors that function under harsh industrial applications. Several sequence-based approaches can generate thermostable variants of mesophilic proteins. To gain insight into the mechanisms by which proteins become more stable, we use structural and dynamic analyses to compare two popular approaches, ancestral sequence reconstruction (ASR) and the consensus method, used to generate thermostable variants of Elongation Factor Thermo-unstable (EF-Tu)...
December 16, 2017: Structure
https://www.readbyqxmd.com/read/29250804/nonredundant-functions-of-arabidopsis-lecrk-v-2-and-lecrk-vii-1-in-controlling-stomatal-immunity-and-jasmonate-mediated-stomatal-closure
#8
Shweta Yekondi, Fu-Chun Liang, Eiji Okuma, Amandine Radziejwoski, Hsien-Wei Mai, Swadhin Swain, Prashant Singh, Mathieu Gauthier, Hsiao-Chiao Chien, Yoshiyuki Murata, Laurent Zimmerli
Stomatal immunity restricts bacterial entry to leaves through the recognition of microbe-associated molecular patterns (MAMPs) by pattern-recognition receptors (PRRs) and downstream abscisic acid and salicylic acid signaling. Through a reverse genetics approach, we characterized the function of the L-type lectin receptor kinase-V.2 (LecRK-V.2) and -VII.1 (LecRK-VII.1). Analyses of interactions with the PRR FLAGELLIN SENSING2 (FLS2) were performed by co-immunoprecipitation and bimolecular fluorescence complementation and whole-cell patch-clamp analyses were used to evaluate guard cell Ca2+ -permeable cation channels...
December 18, 2017: New Phytologist
https://www.readbyqxmd.com/read/29235176/roles-of-elusive-translational-gtpases-come-to-light-and-inform-on-the-process-of-ribosome-biogenesis-in-bacteria
#9
REVIEW
Michelle R Gibbs, Kurt Fredrick
Protein synthesis relies on several translational GTPases (trGTPases), related proteins that couple the hydrolysis of GTP to specific molecular events on the ribosome. Most bacterial trGTPases, including IF2, EF-Tu, EF-G, and RF3, play well-known roles in translation. The cellular functions of LepA (also termed EF4) and BipA (also termed TypA), on the other hand, have remained enigmatic. Recent studies provide compelling in vivo evidence that LepA and BipA function in biogenesis of the 30S and 50S subunit, respectively...
December 13, 2017: Molecular Microbiology
https://www.readbyqxmd.com/read/29155943/logical-engineering-of-d-arm-and-t-stem-of-trna-that-enhances-d-amino-acid-incorporation
#10
Takayuki Katoh, Yoshihiko Iwane, Hiroaki Suga
A bacterial translation factor EF-P alleviates ribosomal stalling caused by polyproline sequence by accelerating Pro-Pro formation. EF-P recognizes a specific D-arm motif found in tRNAPro isoacceptors, 9-nt D-loop closed by a stable D-stem sequence, for Pro-selective peptidyl-transfer acceleration. It is also known that the T-stem sequence on aminoacyl-tRNAs modulates strength of the interaction with EF-Tu, giving enhanced incorporation of non-proteinogenic amino acids such as some N-methyl amino acids. Based on the above knowledge, we logically engineered tRNA's D-arm and T-stem sequences to investigate a series of tRNAs for the improvement of consecutive incorporation of d-amino acids and an α, α-disubstituted amino acid...
November 16, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/29058676/model-based-local-density-sharpening-of-cryo-em-maps
#11
Arjen J Jakobi, Matthias Wilmanns, Carsten Sachse
Atomic models based on high-resolution density maps are the ultimate result of the cryo-EM structure determination process. Here, we introduce a general procedure for local sharpening of cryo-EM density maps based on prior knowledge of an atomic reference structure. The procedure optimizes contrast of cryo-EM densities by amplitude scaling against the radially averaged local falloff estimated from a windowed reference model. By testing the procedure using six cryo-EM structures of TRPV1, β-galactosidase, γ-secretase, ribosome-EF-Tu complex, 20S proteasome and RNA polymerase III, we illustrate how local sharpening can increase interpretability of density maps in particular in cases of resolution variation and facilitates model building and atomic model refinement...
October 23, 2017: ELife
https://www.readbyqxmd.com/read/29045140/the-conformational-change-in-elongation-factor-tu-involves-separation-of-its-domains
#12
Jonathan Lai, Zhaleh Ghaemi, Zaida Luthey-Schulten
Elongation factor Tu (EF-Tu) is a highly conserved GTPase that is responsible for supplying the aminoacylated tRNA to the ribosome. Upon binding to the ribosome, EF-Tu undergoes GTP hydrolysis, which drives a major conformational change, triggering the release of aminoacylated tRNA to the ribosome. Using a combination of molecular simulation techniques, we studied the transition between the pre- and post-hydrolysis structures through two distinct pathways. We show that the transition free energy is minimal along a non-intuitive pathway that involves "separation" of the GTP binding domain (domain 1) from the OB folds (domains 2 and 3), followed by domain 1 rotation, and, eventually, locking the EF-Tu conformation in the post-hydrolysis state...
October 27, 2017: Biochemistry
https://www.readbyqxmd.com/read/29033958/enhanced-bacterial-wilt-resistance-in-potato-through-expression-of-arabidopsis-efr-and-introgression-of-quantitative-resistance-from-solanum-commersonii
#13
Federico Boschi, Claudia Schvartzman, Sara Murchio, Virginia Ferreira, Maria I Siri, Guillermo A Galván, Matthew Smoker, Lena Stransfeld, Cyril Zipfel, Francisco L Vilaró, Marco Dalla-Rizza
Bacterial wilt (BW) caused by Ralstonia solanacearum is responsible for substantial losses in cultivated potato (Solanum tuberosum) crops worldwide. Resistance genes have been identified in wild species; however, introduction of these through classical breeding has achieved only partial resistance, which has been linked to poor agronomic performance. The Arabidopsis thaliana (At) pattern recognition receptor elongation factor-Tu (EF-Tu) receptor (EFR) recognizes the bacterial pathogen-associated molecular pattern EF-Tu (and its derived peptide elf18) to confer anti-bacterial immunity...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/28973472/structure-of-a-novel-antibacterial-toxin-that-exploits-elongation-factor-tu-to-cleave-specific-transfer-rnas
#14
Karolina Michalska, Grant C Gucinski, Fernando Garza-Sánchez, Parker M Johnson, Lucy M Stols, William H Eschenfeldt, Gyorgy Babnigg, David A Low, Celia W Goulding, Andrzej Joachimiak, Christopher S Hayes
Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides...
September 29, 2017: Nucleic Acids Research
https://www.readbyqxmd.com/read/28910101/anisotropic-fluctuations-in-the-ribosome-determine-trna-kinetics
#15
Huan Yang, Jeffrey K Noel, Paul C Whitford
The ribosome is a large ribonucleoprotein complex that is responsible for the production of proteins in all organisms. Accommodation is the process by which an incoming aminoacyl-tRNA (aa-tRNA) molecule binds the ribosomal A site, and its kinetics has been implicated in the accuracy of tRNA selection. In addition to rearrangements in the aa-tRNA molecule, the L11 stalk can undergo large-scale anisotropic motions during translation. To explore the potential impact of this protruding region on the rate of aa-tRNA accommodation, we used molecular dynamics simulations with a simplified model to evaluate the free energy as a function of aa-tRNA position...
October 2, 2017: Journal of Physical Chemistry. B
https://www.readbyqxmd.com/read/28894125/elongation-factor-tu-is-a-multifunctional-and-processed-moonlighting-protein
#16
Michael Widjaja, Kate Louise Harvey, Lisa Hagemann, Iain James Berry, Veronica Maria Jarocki, Benjamin Bernard Armando Raymond, Jessica Leigh Tacchi, Anne Gründel, Joel Ricky Steele, Matthew Paul Padula, Ian George Charles, Roger Dumke, Steven Philip Djordjevic
Many bacterial moonlighting proteins were originally described in medically, agriculturally, and commercially important members of the low G + C Firmicutes. We show Elongation factor Tu (Ef-Tu) moonlights on the surface of the human pathogens Staphylococcus aureus (SaEf-Tu) and Mycoplasma pneumoniae (MpnEf-Tu), and the porcine pathogen Mycoplasma hyopneumoniae (MhpEf-Tu). Ef-Tu is also a target of multiple processing events on the cell surface and these were characterised using an N-terminomics pipeline...
September 11, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28866886/efficient-expression-of-glutathione-peroxidase-with-chimeric-trna-in-amber-less-escherichia-coli
#17
Zhenlin Fan, Jian Song, Tuchen Guan, Xiuxiu Lv, Jingyan Wei
The active center of selenium-containing glutathione peroxidase (GPx) is selenocysteine (Sec), which is is biosynthesized on its tRNA in organisms. The decoding of Sec depends on a specific elongation factor and a Sec Insertion Sequence (SECIS) to suppress the UGA codon. The expression of mammalian GPx is extremely difficult with traditional recombinant DNA technology. Recently, a chimeric tRNA (tRNA(UTu)) that is compatible with elongation factor Tu (EF-Tu) has made selenoprotein expression easier. In this study, human glutathione peroxidase (hGPx) was expressed in amber-less Escherichia coli C321...
September 12, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28865219/expression-and-immunological-evaluation-of-elongation-factor-tu-of-streptococcus-suis-serotype-2
#18
X J Xia, L Wang, L K Cheng, Z Q Shen, S G Li, J L Wang
Streptococcus suis serotype 2 (SS2) is considered as a major pathogen that causes sepsis and meningitis in piglets and humans, but knowledge of its antigenic proteins remains limited so far. The surface-related proteins of pathogens often play significant roles in bacterium-host interactions and infection. Here, we obtained the elongation factor Tu (EF-Tu) gene of Streptococcus suis and constructed the recombinant expression plasmid successfully. The target recombinant plasmid was then expressed in Escherichia coli and the immuno-protection of the recombinant protein was subsequently evaluated as well...
March 1, 2017: Polish Journal of Veterinary Sciences
https://www.readbyqxmd.com/read/28865072/gallibacterium-elongation-factor-tu-possesses-amyloid-like-protein-characteristics-participates-in-cell-adhesion-and-is-present-in-biofilms
#19
Jaqueline López-Ochoa, J Fernando Montes-García, Candelario Vázquez, Patricia Sánchez-Alonso, Victor M Pérez-Márquez, Patrick J Blackall, Sergio Vaca, Erasmo Negrete-Abascal
Gallibacterium, which is a bacterial pathogen in chickens, can form biofilms. Amyloid proteins present in biofilms bind Congo red dye. The aim of this study was to characterize the cell-surface amyloid-like protein expressed in biofilms formed by Gallibacterium strains and determine the relationship between this protein and curli, which is an amyloid protein that is commonly expressed by members of the Enterobacteriaceae family. The presence of amyloid-like proteins in outer membrane protein samples from three strains of G...
September 2017: Journal of Microbiology / the Microbiological Society of Korea
https://www.readbyqxmd.com/read/28851849/functional-constraints-on-replacing-an-essential-gene-with-its-ancient-and-modern-homologs
#20
Betül Kacar, Eva Garmendia, Nurcan Tuncbag, Dan I Andersson, Diarmaid Hughes
Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms...
August 29, 2017: MBio
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