Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Altered oligomerization properties of N316 mutants of Escherichia coli TyrR.

ABSTRACT: The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrR(E274Q), a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrR(N316D) and TyrR(N316R) mutants. Gel filtration analyses revealed that the ligand-mediated oligomer formation was enhanced and diminished for TyrR(N316D) and TyrR(N316R), respectively, compared with the wild-type TyrR. ADP was substituted for ATP in the oligomer formation of TyrR(N316D).

SUBMITTER: Koyanagi T

PROVIDER: S-EPMC2593242 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Altered oligomerization properties of N316 mutants of Escherichia coli TyrR.

Koyanagi Takashi T Katayama Takane T Suzuki Hideyuki H Kumagai Hidehiko H

Journal of bacteriology 20081017 24

The transcriptional regulator TyrR is known to undergo a dimer-to-hexamer conformational change in response to aromatic amino acids, through which it controls gene expression. In this study, we identified N316D as the second-site suppressor of Escherichia coli TyrR(E274Q), a mutant protein deficient in hexamer formation. N316 variants exhibited altered in vivo regulatory properties, and the most drastic changes were observed for TyrR(N316D) and TyrR(N316R) mutants. Gel filtration analyses reveal ...[more]

PMID: 18931124

Similar Datasets

Characterization of Escherichia coli type 1 pilus mutants with altered binding specificities.

Project description:PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.

| S-EPMC95295 | biostudies-literature

The oligomerization of CynR in Escherichia coli.

Project description:Deletion analysis and alanine-scanning based on a homology-based interaction model were used to identify determinants of oligomerization in the transcriptional regulator CynR, a member of the LysR-type transcriptional regulator (LTTR) family. Deletion analysis confirmed that the putative regulatory domain of CynR was essential for driving the oligomerization of lambda repressor-CynR fusion proteins. The interaction surface of a different LTTR and OxyR was mapped onto a multiple sequence alignment of the LTTR family. This mapping identified putative contacts in the CynR regulatory domain dimer interface, which were targeted for alanine-scanning mutagenesis. Oligomerization was assayed by the ability of mutant lambda repressor-CynR fusions to assemble in E. coli revealing interesting similarities and differences between OxyR and CynR.

| S-EPMC2788285 | biostudies-literature

The oligomerization of OxyR in Escherichia coli.

Project description:We examine the contribution of residues at the dimer interface of the transcriptional regulator OxyR to oligomerization. Residues in contact across the dimer interface of OxyR were identified using the program Quaternary Contacts (QContacts). Site-directed mutagenesis was performed on the non-alanine or glycine residues identified in the resultant contact profile and the oligomerization ability of the mutant proteins was tested using the lambdacI repressor system to identify residues that are hot spots in OxyR. We compared the properties of these hot spots to those described in the literature from other systems. The hot spots identified in this study are not especially conserved amongst a set of OxyR orthologs.

| S-EPMC2708027 | biostudies-literature

Flavodoxin mutants of Escherichia coli K-12.

Project description:The flavodoxins are flavin mononucleotide-containing electron transferases. Flavodoxin I has been presumed to be the only flavodoxin of Escherichia coli, and its gene, fldA, is known to belong to the soxRS (superoxide response) oxidative stress regulon. An insertion mutation of fldA was constructed and was lethal under both aerobic and anaerobic conditions; only cells that also had an intact (fldA(+)) allele could carry it. A second flavodoxin, flavodoxin II, was postulated, based on the sequence of its gene, fldB. Unlike the fldA mutant, an fldB insertion mutant is a viable prototroph in the presence or absence of oxygen. A high-copy-number fldB(+) plasmid did not complement the fldA mutation. Therefore, there must be a vital function for which FldB cannot substitute for flavodoxin I. An fldB-lacZ fusion was not induced by H(2)O(2) and is therefore not a member of the oxyR regulon. However, it displayed a soxS-dependent induction by paraquat (methyl viologen), and the fldB gene is preceded by two overlapping regions that resemble known soxS binding sites. The fldB insertion mutant did not have an increased sensitivity to the effects of paraquat on either cellular viability or the expression of a soxS-lacZ fusion. Therefore, fldB is a new member of the soxRS (superoxide response) regulon, a group of genes that is induced primarily by univalent oxidants and redox cycling compounds. However, the reactions in which flavodoxin II participates and its role during oxidative stress are unknown.

| S-EPMC101859 | biostudies-literature

Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules.

Project description:Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

| S-EPMC2816605 | biostudies-literature

Escherichia coli mutants induced by multi-ion irradiation.

Project description:Wild-type Escherichia coli K12 strain W3110 was irradiated by 10 keV nitrogen ions. Specifically, irradiation was performed six times by N(+) ions, followed by the selection of lac constitutive mutants, and each time a stable S55 mutant was produced. By sequencing the whole genome, the fine map of S55 was completed. Compared with reference sequences, a total of eighteen single nucleotide polymorphisms (SNPs), two insertions and deletions (Indels), and nine structural variations (SVs) were found in the S55 genome. Among the 18 SNPs, 11 are transversional from A, T or C to G, accounting for 55.6% of point mutations. GCCA insertion occurs in the target gene lacI. Four SNPs, including three in rlpB and one in ygbN, are connected with cell envelope and transport. All nine structural variations of S55 are deletions and contain insertion sequence (IS) elements. Six deleted SVs contain disrupted ISs, nonfunctional pseudogenes, and one more 23 252 bp SV in the Rac prophage region. Overall, our results show that deletion bias observed in E. coli K12 genome evolution is generally related to the deletion of some nonfunctional regions. Furthermore, since ISs are unstable factors in a genome, the multi-ion irradiations that caused these deleted fragments in S55 turn out to be beneficial to genome stability, generating a wider mutational spectrum. Thus, it is possible that the mutation of these genes increases the ability of the E. coli genome to resist etch and damage caused by ion irradiation.

| S-EPMC3483858 | biostudies-literature

Isolation of SOS constitutive mutants of Escherichia coli.

Project description:The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.

| S-EPMC523190 | biostudies-literature

Cadmium toxicity in glutathione mutants of Escherichia coli.

Project description:The higher affinity of Cd(2+) for sulfur compounds than for nitrogen and oxygen led to the theoretical consideration that cadmium toxicity should result mainly from the binding of Cd(2+) to sulfide, thiol groups, and sulfur-rich complex compounds rather than from Cd(2+) replacement of transition-metal cations from nitrogen- or oxygen-rich biological compounds. This hypothesis was tested by using Escherichia coli for a global transcriptome analysis of cells synthesizing glutathione (GSH; wild type), gamma-glutamylcysteine (DeltagshB mutant), or neither of the two cellular thiols (DeltagshA mutant). The resulting data, some of which were validated by quantitative reverse transcription-PCR, were sorted using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system, which groups genes hierarchically with respect to the cellular functions of their respective products. The main difference among the three strains concerned tryptophan biosynthesis, which was up-regulated in wild-type cells upon cadmium shock and strongly up-regulated in DeltagshA cells but repressed in DeltagshB cells containing gamma-glutamylcysteine instead of GSH. Overall, however, all three E. coli strains responded to cadmium shock similarly, with the up-regulation of genes involved in protein, disulfide bond, and oxidative damage repair; cysteine and iron-sulfur cluster biosynthesis; the production of proteins containing sensitive iron-sulfur clusters; the storage of iron; and the detoxification of Cd(2+) by efflux. General energy conservation pathways and iron uptake were down-regulated. These findings indicated that the toxic action of Cd(2+) indeed results from the binding of the metal cation to sulfur, lending support to the hypothesis tested.

| S-EPMC2493267 | biostudies-literature

Flavin-induced oligomerization in Escherichia coli adaptive response protein AidB.

Project description:The process known as "adaptive response" allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity for flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 ± 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 Å resolution crystal structure in space group P3(2) that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.

| S-EPMC3217306 | biostudies-literature

A genetic analysis of various functions of the TyrR protein of Escherichia coli.

Project description:The TyrR protein is involved in both repression and activation of the genes of the TyrR regulon. Correction of an error in a previously published sequence has revealed a Cro-like helix-turn-helix DNA-binding domain near the carboxyl terminus. Site-directed mutagenesis in this region has generated a number of mutants that can no longer repress or activate. Deletions of amino acid residues 5 to 42 produced a protein that could repress but not activate. The central domain of TyrR contains an ATP-binding site and is homologous with the NtrC family of activator proteins. A mutation to site A of the ATP-binding site and other mutations in this region affect tyrosine-mediated repression but do not prevent activation or phenylalanine-mediated repression of aroG.

| S-EPMC203971 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data