scholarly journals Structure of Crystallined-Tyr-tRNATyrDeacylase

2001 ◽  
Vol 276 (50) ◽  
pp. 47285-47290 ◽  
Author(s):  
Maria-Laura Ferri-Fioni ◽  
Emmanuelle Schmitt ◽  
Julie Soutourina ◽  
Pierre Plateau ◽  
Yves Mechulam ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Active Sites ◽  
Yeast Cells ◽  
Crystallographic Structure ◽  
Cell Growth Inhibition ◽  
E Coli ◽  
Sequence Homologies ◽  
Β Sheet

Cell growth inhibition by severald-amino acids can be explained by anin vivoproduction ofd-aminoacyl-tRNA molecules.Escherichia coliand yeast cells express an enzyme,d-Tyr-tRNATyrdeacylase, capable of recycling suchd-aminoacyl-tRNA molecules into free tRNA andd-amino acid. Accordingly, upon inactivation of the genes of the above deacylases, the toxicity ofd-amino acids increases. Orthologs of the deacylase are found in many cells. In this study, the crystallographic structure of dimericE. colid-Tyr-tRNATyrdeacylase at 1.55 Å resolution is reported. The structure corresponds to a β-barrel closed on one side by a β-sheet lid. This barrel results from the assembly of the two subunits. Analysis of the structure in relation with sequence homologies in the orthologous family suggests the location of the active sites at the carboxy end of the β-strands. The solved structure markedly differs from those of all other documented tRNA-dependent hydrolases.

scholarly journals A nucleotide-dependent oligomerization of the Escherichia coli replication initiator DnaA requires residue His136 for remodeling of the chromosomal origin

2019 ◽  
Author(s):  
Rahul Saxena ◽  
Christopher B Stanley ◽  
Pankaj Kumar ◽  
Matthew J Cuneo ◽  
Digvijay Patil ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Imidazole Ring ◽  
Crystallographic Structure ◽  
Aquifex Aeolicus ◽  
E Coli ◽  
Initiator Protein ◽  
Functional Studies ◽  
High Affinity ◽  
Replication Initiator

Abstract Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP–PNP–DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP–PNP–DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.

scholarly journals Substrate specificities of Escherichia coli ItaT that acetylates aminoacyl-tRNAs

2020 ◽  
Author(s):  
Chuqiao Zhang ◽  
Yuka Yashiro ◽  
Yuriko Sakaguchi ◽  
Tsutomu Suzuki ◽  
Kozo Tomita
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Group ◽  
Hydrophobic Pocket ◽  
Hydrophobic Residues ◽  
Substrate Specificities ◽  
E Coli ◽  
Hydrophobic Amino Acids

Abstract Escherichia coli ItaT toxin reportedly acetylates the α-amino group of the aminoacyl-moiety of Ile-tRNAIle specifically, using acetyl-CoA as an acetyl donor, thereby inhibiting protein synthesis. The mechanism of the substrate specificity of ItaT had remained elusive. Here, we present functional and structural analyses of E. coli ItaT, which revealed the mechanism of ItaT recognition of specific aminoacyl-tRNAs for acetylation. In addition to Ile-tRNAIle, aminoacyl-tRNAs charged with hydrophobic residues, such as Val-tRNAVal and Met-tRNAMet, were acetylated by ItaT in vivo. Ile-tRNAIle, Val-tRNAVal and Met-tRNAMet were acetylated by ItaT in vitro, while aminoacyl-tRNAs charged with other hydrophobic residues, such as Ala-tRNAAla, Leu-tRNALeu and Phe-tRNAPhe, were less efficiently acetylated. A comparison of the structures of E. coli ItaT and the protein N-terminal acetyltransferase identified the hydrophobic residues in ItaT that possibly interact with the aminoacyl moiety of aminoacyl-tRNAs. Mutations of the hydrophobic residues of ItaT reduced the acetylation activity of ItaT toward Ile-tRNAIlein vitro, as well as the ItaT toxicity in vivo. Altogether, the size and shape of the hydrophobic pocket of ItaT are suitable for the accommodation of the specific aminoacyl-moieties of aminoacyl-tRNAs, and ItaT has broader specificity toward aminoacyl-tRNAs charged with certain hydrophobic amino acids.

RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

scholarly journals RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli.

1987 ◽  
Vol 7 (3) ◽  
pp. 1180-1192 ◽  
Author(s):  
R Fleer ◽  
C M Nicolet ◽  
G A Pure ◽  
E C Friedberg
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Insertional Mutagenesis ◽  
Genomic Library ◽  
Excision Repair ◽  
Essential Gene ◽  
Yeast Cells ◽  
E Coli ◽  
Uv Sensitivity

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

scholarly journals Characterization and in Vivo Cloning of prlC, a Suppressor of Signal Sequence Mutations in Escherichia coli K12

Genetics ◽  
1987 ◽  
Vol 116 (4) ◽  
pp. 513-521
Author(s):  
Nancy J Trun ◽  
Thomas J Silhavy
Keyword(s):  
Escherichia Coli ◽  
Genetic Mapping ◽  
Signal Sequence ◽  
Illegitimate Recombination ◽  
Mutant Phenotype ◽  
E Coli ◽  
Physical Location ◽  
Sequence Deletion ◽  
New Alleles

ABSTRACT The prlC gene of E. coli was originally identified as an allele, prlC1, which suppresses certain signal sequence mutations in the genes for several exported proteins. We have isolated six new alleles of prlC that also confer this phenotype. These mutations can be placed into three classes based on the degree to which they suppress the lamBsignal sequence deletion, lamBs78. Genetic mapping reveals that the physical location of the mutations in prlC correlates with the strength of the suppression, suggesting that different regions of the gene can be altered to yield a suppressor phenotype. We also describe an in vivo cloning procedure using λplacMu9H. The procedure relies on transposition and illegitimate recombination to generate a specialized transducing phage that carries prlC1. This method should be applicable to any gene for which there is a mutant phenotype.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

scholarly journals Mechanistic insights into synergy between nalidixic acid and tetracycline against clinical isolates of Acinetobacter baumannii and Escherichia coli

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Amit Gaurav ◽  
Varsha Gupta ◽  
Sandeep K. Shrivastava ◽  
Ranjana Pathania
Keyword(s):  
Escherichia Coli ◽  
Acinetobacter Baumannii ◽  
Nalidixic Acid ◽  
Bacterial Infections ◽  
Clinical Isolates ◽  
Hospital Environment ◽  
Infection Model ◽  
Drug Resistant ◽  
E Coli

AbstractThe increasing prevalence of antimicrobial resistance has become a global health problem. Acinetobacter baumannii is an important nosocomial pathogen due to its capacity to persist in the hospital environment. It has a high mortality rate and few treatment options. Antibiotic combinations can help to fight multi-drug resistant (MDR) bacterial infections, but they are rarely used in the clinics and mostly unexplored. The interaction between bacteriostatic and bactericidal antibiotics are mostly reported as antagonism based on the results obtained in the susceptible model laboratory strain Escherichia coli. However, in the present study, we report a synergistic interaction between nalidixic acid and tetracycline against clinical multi-drug resistant A. baumannii and E. coli. Here we provide mechanistic insight into this dichotomy. The synergistic combination was studied by checkerboard assay and time-kill curve analysis. We also elucidate the mechanism behind this synergy using several techniques such as fluorescence spectroscopy, flow cytometry, fluorescence microscopy, morphometric analysis, and real-time polymerase chain reaction. Nalidixic acid and tetracycline combination displayed synergy against most of the MDR clinical isolates of A. baumannii and E. coli but not against susceptible isolates. Finally, we demonstrate that this combination is also effective in vivo in an A. baumannii/Caenorhabditis elegans infection model (p < 0.001)

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 degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

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