scholarly journals A Rationally Designed Aminoacyl-tRNA Synthetase for Genetically Encoded Fluorescent Amino Acids

2016 ◽  
Author(s):  
Ximena Steinberg ◽  
Jason Galpin ◽  
Gibran Nasir ◽  
Jose Sepulveda-Ugarte ◽  
Romina V. Sepúlveda ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Structure ◽  
Structure Function ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
E Coli ◽  
Promising Strategy ◽  
Fluorescent Amino Acids

AbstractThe incorporation of non-canonical amino acids into proteins has emerged as a promising strategy to manipulate and study protein structure-function relationships with superior precision in vitro and in vivo. To date, fluorescent non-canonical amino acids (f-ncAA) have been successfully incorporated in proteins expressed in bacterial systems, Xenopus oocytes, and HEK-293T cells. Here, we describe the rational generation of an orthogonal aminoacyltRNA synthetase based on the E. coli tyrosine synthetase that is capable of encoding the f-ncAA tyr-coumarin in HEK-293T cells.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 473 ◽  
Author(s):  
Takuya Umehara ◽  
Saori Kosono ◽  
Dieter Söll ◽  
Koji Tamura
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Lysine Acetylation ◽  
Trna Synthetases ◽  
E Coli ◽  
Domains Of Life ◽  
The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

scholarly journals Structure/Function Analysis of the Vaccinia Virus F18 Phosphoprotein, an Abundant Core Component Required for Virion Maturation and Infectivity

2010 ◽  
Vol 84 (13) ◽  
pp. 6846-6860 ◽  
Author(s):  
Nadi T. Wickramasekera ◽  
Paula Traktman
Keyword(s):  
Amino Acids ◽  
Structure Function ◽  
Protein Interactions ◽  
Function Analysis ◽  
Aromatic Amino Acids ◽  
Protein Protein Interactions ◽  
Core Component ◽  
Virion Morphogenesis

ABSTRACT Poxvirus virions, whose outer membrane surrounds two lateral bodies and a core, contain at least 70 different proteins. The F18 phosphoprotein is one of the most abundant core components and is essential for the assembly of mature virions. We report here the results of a structure/function analysis in which the role of conserved cysteine residues, clusters of charged amino acids and clusters of hydrophobic/aromatic amino acids have been assessed. Taking advantage of a recombinant virus in which F18 expression is IPTG (isopropyl-β-d-thiogalactopyranoside) dependent, we developed a transient complementation assay to evaluate the ability of mutant alleles of F18 to support virion morphogenesis and/or to restore the production of infectious virus. We have also examined protein-protein interactions, comparing the ability of mutant and WT F18 proteins to interact with WT F18 and to interact with the viral A30 protein, another essential core component. We show that F18 associates with an A30-containing multiprotein complex in vivo in a manner that depends upon clusters of hydrophobic/aromatic residues in the N′ terminus of the F18 protein but that it is not required for the assembly of this complex. Finally, we confirmed that two PSSP motifs within F18 are the sites of phosphorylation by cellular proline-directed kinases in vitro and in vivo. Mutation of both of these phosphorylation sites has no apparent impact on virion morphogenesis but leads to the assembly of virions with significantly reduced infectivity.

scholarly journals Fusion of the N-terminal 119 amino acids with the RelA-CTD renders its growth inhibitory effects (p)ppGpp-dependent

2021 ◽  
Author(s):  
Jayashree Pohnerkar ◽  
Krishma Tailor ◽  
Prarthi Sagar ◽  
Keyur Dave
Keyword(s):  
Amino Acids ◽  
Feedback Regulation ◽  
Inhibitory Effects ◽  
E Coli ◽  
Growth Inhibitory ◽  
In The Wild ◽  
Regulatory Aspect ◽  
And Function

The guanosine nucleotide derivatives ppGpp and pppGpp are central to the remarkable capacity of bacteria to adapt to fluctuating environment and metabolic perturbations. These alarmones are synthesized by two proteins, RelA and SpoT in E. coli and the activities of each of the two enzymes are highly regulated for homeostatic control of (p)ppGpp levels in the cell. Although the domain structure and function of RelA are well defined, the findings of this study unfold the regulatory aspect of RelA that is possibly relevant in vivo. We uncover here the importance of the N-terminal 1-119 amino acids of the enzymatically compromised (p)ppGpp hydrolytic domain (HD) of monofunctional RelA for the (p)ppGpp mediated regulation of RelA-CTD function. We find that even moderate level expression of RelA appreciably reduces growth when the basal levels of (p)ppGpp in the cells are higher than in the wild type, an effect independent of its ability to synthesize (p)ppGpp. This is evidenced by the growth inhibitory effects of oversynthesis of the RelA-CTD in the relA+ strain but not in relA null mutant, suggesting the requirement of the functional RelA protein for basal level synthesis of (p)ppGpp, accordingly corroborated by the restoration of the growth inhibitory effects of the RelA-CTD expression in the relA1 spoT202 mutant. The N-terminal 119 amino acids of RelA fused in-frame with the RelA-CTD, both from 406-744 amino acids (including TGS) and from 454-744 amino acids (sans TGS) caused growth inhibition only in spoT1 and spoT202 relA1 mutants, uncovering the hitherto unrealized (p)ppGpp-dependent regulation of RelA-CTD function. An incremental rise in the (p)ppGpp levels is proposed to progressively modulate the interaction of RelA-CTD with the ribosomes, with possible implications in the feedback regulation of the N-terminal (p)ppGpp synthesis function, a proposal that best explains the nonlinear relationship between (p)ppGpp synthesis and increased ratio of RelA:ribosomes, both in vitro as well as in vivo.

scholarly journals The relA/spoT-Homologous Gene inStreptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

1998 ◽  
Vol 180 (16) ◽  
pp. 4123-4132 ◽  
Author(s):  
Oscar H. Martínez-Costa ◽  
Miguel A. Fernández-Moreno ◽  
Francisco Malpartida
Keyword(s):  
Amino Acids ◽  
Deletion Mutant ◽  
Streptomyces Coelicolor ◽  
Stringent Response ◽  
Morphological Differentiation ◽  
Antibiotic Biosynthesis ◽  
Homologous Gene ◽  
E Coli

ABSTRACT Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined.S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence thatS. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of anS. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. colitruncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, arel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition,E. coli relA in an S. coelicolor reldeletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-typerel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.

scholarly journals Molecular characterization of the TonB2 protein from the fish pathogen Vibrio anguillarum

2009 ◽  
Vol 418 (1) ◽  
pp. 49-59 ◽  
Author(s):  
Claudia S. López ◽  
R. Sean Peacock ◽  
Jorge H. Crosa ◽  
Hans J. Vogel
Keyword(s):  
Amino Acids ◽  
Solution Structure ◽  
Bacterial Species ◽  
Vibrio Anguillarum ◽  
Fish Pathogen ◽  
E Coli ◽  
Terminal Domain ◽  
C Terminus

In the fish pathogen Vibrio anguillarum the TonB2 protein is essential for the uptake of the indigenous siderophore anguibactin. Here we describe deletion mutants and alanine replacements affecting the final six amino acids of TonB2. Deletions of more than two amino acids of the TonB2 C-terminus abolished ferric-anguibactin transport, whereas replacement of the last three residues resulted in a protein with wild-type transport properties. We have solved the high-resolution solution structure of the TonB2 C-terminal domain by NMR spectroscopy. The core of this domain (residues 121–206) has an αββαβ structure, whereas residues 76–120 are flexible and extended. This overall folding topology is similar to the Escherichia coli TonB C-terminal domain, albeit with two differences: the β4 strand found at the C-terminus of TonB is absent in TonB2, and loop 3 is extended by 9 Å (0.9 nm) in TonB2. By examining several mutants, we determined that a complete loop 3 is not essential for TonB2 activity. Our results indicate that the β4 strand of E. coli TonB is not required for activity of the TonB system across Gram-negative bacterial species. We have also determined, through NMR chemical-shift-perturbation experiments, that the E. coli TonB binds in vitro to the TonB box from the TonB2-dependent outer membrane transporter FatA; moreover, it can substitute in vivo for TonB2 during ferric-anguibactin transport in V. anguillarum. Unexpectedly, TonB2 did not bind in vitro to the FatA TonB-box region, suggesting that additional factors may be required to promote this interaction. Overall our results indicate that TonB2 is a representative of a different class of TonB proteins.

scholarly journals Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

1978 ◽  
Vol 176 (1) ◽  
pp. 305-318 ◽  
Author(s):  
Julia Hughes ◽  
Graham Mellows
Keyword(s):  
Rna Synthesis ◽  
Inhibitory Effect ◽  
Trna Synthetase ◽  
Threonine Deaminase ◽  
Trna Synthetases ◽  
E Coli ◽  
Naturally Occurring ◽  
Pseudomonic Acid

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RCrel, whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNAIle. Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.

scholarly journals A human pathology-related mutation prevents import of an aminoacyl-tRNA synthetase into mitochondria

2011 ◽  
Vol 433 (3) ◽  
pp. 441-446 ◽  
Author(s):  
Marie Messmer ◽  
Catherine Florentz ◽  
Hagen Schwenzer ◽  
Gert C. Scheper ◽  
Marjo S. van der Knaap ◽  
...  
Keyword(s):  
Nuclear Gene ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Mitochondrial Translation ◽  
Gene Coding ◽  
Human Pathology ◽  
Key Enzyme ◽  
Targeting Signal

Mutations in the nuclear gene coding for the mitochondrial aspartyl-tRNA synthetase, a key enzyme for mitochondrial translation, are correlated with leukoencephalopathy. A Ser45 to Gly45 mutation is located in the predicted targeting signal of the protein. We demonstrate in the present study, by in vivo and in vitro approaches, that this pathology-related mutation impairs the import process across mitochondrial membranes.

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