Purification and characterization of a 59-kilodalton protein that specifically binds to NF-kappa B-binding motifs of the defense protein genes of Sarcophaga peregrina (the flesh fly).

Various Sarcophaga peregrina (flesh fly) defense protein genes were shown to be activated when NIH-Sape-4 cells were cultured with bacterial lipopolysaccharides or beta-1,3-glucan. The 5' upstream regions of the defense protein genes were found to have common motifs showing similarity to the mammalian NF-kappa B-binding consensus sequence. A protein with affinity to the NF-kappa B-binding motif of the Sarcophaga lectin promoter was identified and purified to near homogeneity. This 59-kDa protein also bound to the NF-kappa B-binding motifs of other defense protein genes, e.g., sarcotoxin I and sarcotoxin II genes. This protein was found in both the cytoplasmic and the nuclear fractions of the cells, and it appeared to migrate from the cytoplasm to the nucleus on treatment of the cells with lipopolysaccharides. This 59-kDa protein is probably a transcriptional regulator of the genes for defense proteins of S. peregrina.

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An SOS Regulon under Control of a Noncanonical LexA-Binding Motif in the Betaproteobacteria

Journal of Bacteriology ◽

10.1128/jb.00035-15 ◽

2015 ◽

Vol 197 (16) ◽

pp. 2622-2630 ◽

Cited By ~ 6

Author(s):

Neus Sanchez-Alberola ◽

Susana Campoy ◽

David Emerson ◽

Jordi Barbé ◽

Ivan Erill

Keyword(s):

In Silico ◽

Transcriptional Regulatory Network ◽

Sequence Similarity ◽

Consensus Sequence ◽

Binding Motif ◽

Binding Motifs ◽

Content Type ◽

Lexa Protein ◽

Lexa Binding

ABSTRACTThe SOS response is a transcriptional regulatory network governed by the LexA repressor that activates in response to DNA damage. In theBetaproteobacteria, LexA is known to target a palindromic sequence with the consensus sequence CTGT-N8-ACAG. We report the characterization of a LexA regulon in the iron-oxidizing betaproteobacteriumSideroxydans lithotrophicus.In silicoandin vitroanalyses show that LexA targets six genes by recognizing a binding motif with the consensus sequence GAACGaaCGTTC, which is strongly reminiscent of theBacillus subtilisLexA-binding motif. We confirm that the closely relatedGallionella capsiferriformansshares the same LexA-binding motif, andin silicoanalyses indicate that this motif is also conserved in theNitrosomonadalesand theMethylophilales. Phylogenetic analysis of LexA and the alpha subunit of DNA polymerase III (DnaE) reveal that the organisms harboring this noncanonical LexA form a compact taxonomic cluster within theBetaproteobacteria. However, theirlexAgene is unrelated to the standardBetaproteobacterialexA, and there is evidence of its spread through lateral gene transfer. In contrast to other reported cases of noncanonical LexA-binding motifs, the regulon ofS. lithotrophicusis comparable in size and function to that of many otherBetaproteobacteria, suggesting that a convergent SOS regulon has reevolved under the control of a new LexA protein. Analysis of the DNA-binding domain ofS. lithotrophicusLexA reveals little sequence similarity with that of other LexA proteins targeting similar binding motifs, suggesting that network structure may limit site evolution or that structural constrains make theB. subtilis-type motif an optimal interface for multiple LexA sequences.IMPORTANCEUnderstanding the evolution of transcriptional systems enables us to address important questions in microbiology, such as the emergence and transfer potential of different regulatory systems to regulate virulence or mediate responses to stress. The results reported here constitute the first characterization of a noncanonical LexA protein regulating a standard SOS regulon. This is significant because it illustrates how a complex transcriptional program can be put under the control of a novel transcriptional regulator. Our results also reveal a substantial degree of plasticity in the LexA recognition domain, raising intriguing questions about the space of protein-DNA interfaces and the specific evolutionary constrains faced by these elements.

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Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.3002-3014.1993 ◽

1993 ◽

Vol 13 (5) ◽

pp. 3002-3014

Author(s):

K Kudrycki ◽

C Stein-Izsak ◽

C Behn ◽

M Grillo ◽

R Akeson ◽

...

Keyword(s):

Nuclear Protein ◽

Consensus Sequence ◽

Binding Motif ◽

Sequence Motif ◽

Olfactory Neuron ◽

Sequence Motifs ◽

Mobility Shift ◽

Specific Expression ◽

Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

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Olf-1-binding site: characterization of an olfactory neuron-specific promoter motif.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.3002 ◽

1993 ◽

Vol 13 (5) ◽

pp. 3002-3014 ◽

Cited By ~ 74

Author(s):

K Kudrycki ◽

C Stein-Izsak ◽

C Behn ◽

M Grillo ◽

R Akeson ◽

...

Keyword(s):

Nuclear Protein ◽

Consensus Sequence ◽

Binding Motif ◽

Sequence Motif ◽

Olfactory Neuron ◽

Sequence Motifs ◽

Mobility Shift ◽

Specific Expression ◽

Mobility Shift Assay

We report characterization of several domains within the 5' flanking region of the olfactory marker protein (OMP) gene that may participate in regulating transcription of this and other olfactory neuron-specific genes. Analysis by electrophoretic mobility shift assay and DNase I footprinting identifies two regions that contain a novel sequence motif. Interactions between this motif and nuclear proteins were detected only with nuclear protein extracts derived from olfactory neuroepithelium, and this activity is more abundant in olfactory epithelium enriched in immature neurons. We have designated a factor(s) involved in this binding as Olf-1. The Olf-1-binding motif consensus sequence was defined as TCCCC(A/T)NGGAG. Studies with transgenic mice indicate that a 0.3-kb fragment of the OMP gene containing one Olf-1 motif is sufficient for olfactory tissue-specific expression of the reporter gene. Some of the other identified sequence motifs also interact specifically with olfactory nuclear protein extracts. We propose that Olf-1 is a novel, olfactory neuron-specific trans-acting factor involved in the cell-specific expression of OMP.

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A homolog of human transcription factor NF-X1 encoded by the Drosophila shuttle craft gene is required in the embryonic central nervous system.

Molecular and Cellular Biology ◽

10.1128/mcb.16.1.192 ◽

1996 ◽

Vol 16 (1) ◽

pp. 192-201 ◽

Cited By ~ 27

Author(s):

N D Stroumbakis ◽

Z Li ◽

P P Tolias

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Transcription Factor ◽

Consensus Sequence ◽

Preliminary Evidence ◽

Phenotypic Characterization ◽

Binding Motif ◽

New Family

NF-X1 is a novel cytokine-inducible transcription factor that has been implicated in the control of immune responses in humans, presumably by regulating expression of class II major histocompatibility genes. Here we report the cloning and genetic characterization of the first reported NF-X1 homolog, which is encoded by the Drosophila melanogaster shuttle craft (stc) gene. The deduced sequence of the fly and human proteins defines a new family of molecules distinguished by a novel cysteine-rich DNA-binding motif (consisting of seven copies of the consensus sequence Cx3Cx3LxCGx0-5HxCx3CHxGxCx2Cx7-9CxC). We have identified and begun a phenotypic characterization of mutations in the stc gene. stc mutants die at the end of embryogenesis, when they appear to be incapable of coordinating the typical peristaltic contraction waves normally required for embryos to hatch into feeding first instar larvae. Preliminary evidence indicates that the resulting lethality of this behavioral defect is accompanied by subtle morphological abnormalities in the central nervous system, where in wild-type embryos, STC protein is normally localized in the nuclei of repeated cell clusters within each neuromere and brain lobe. Thus, the NF-X1 homolog encoded by the Drosophila stc gene defines a new family of putative transcription factors and plays an essential role in the completion of embryonic development. This study presents the first in vivo genetic analysis of a member of this new protein family.

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A Polymorphism in Thrombospondin-1 Associated with Familial Premature Coronary Artery Disease Alters Ca2+ Binding

Journal of Biological Chemistry ◽

10.1074/jbc.m409632200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 51915-51922 ◽

Cited By ~ 25

Author(s):

Blue-leaf A. Hannah ◽

Tina M. Misenheimer ◽

Michelle M. Pranghofer ◽

Deane F. Mosher

Keyword(s):

Coronary Artery Disease ◽

Coronary Artery ◽

Binding Site ◽

Equilibrium Dialysis ◽

Consensus Sequence ◽

Binding Motif ◽

Premature Coronary Artery Disease ◽

Binding Motifs ◽

Thrombospondin 1 ◽

Artery Disease

A single nucleotide polymorphism that results in substitution at residue 700 of a serine (Ser-700) for an asparagine (Asn-700) in thrombospondin-1 is associated with familial premature coronary artery disease. The polymorphism is located in the first of 13 Ca2+-binding motifs, within a consensus sequence in which Asn-700 likely coordinates Ca2+. Equilibrium dialysis of constructs comprised of the adjoining epidermal growth factor-like module and the Ca2+-binding region (E3Ca) demonstrated that E3Ca Ser-700 binds significantly less Ca2+than E3Ca Asn-700 at low [Ca2+]. The hypothesis that this difference is due to loss of a binding site in Ser-700 protein was tested with truncations of E3Ca containing four (Tr4), three (Tr3), two (Tr2), or one (Tr1) N-terminal Ca2+-binding motifs. The Ser-700 truncation constructs bound 1 fewer Ca2+than matching Asn-700 constructs and exhibited decreased binding affinities. Intrinsic fluorescence of a tryptophan at residue 698 (Trp-698) in the most N-terminal motif was cooperatively quenched by the addition of Ca2+to Asn-700 Tr2, Tr3, and Tr4 constructs. In Ser-700 constructs, quenching of Trp-698 was incomplete in the Tr2 and Tr3 constructs and complete only in the Tr4 construct. Ca2+-induced quenching of Ser-700 constructs required higher [Ca2+] and was slower as shown in stopped-flow experiments than quenching of Asn-700 constructs. Such differences were not found with Tb3+, which quenched the fluorescence of Asn-700 and Ser-700 constructs equivalently. Thus, the Ser-700 polymorphism alters a rapidly filled, high affinity Ca2+-binding site in the first Ca2+-binding motif. Slower Ca2+binding to adjoining motifs partly compensates for the change.

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CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.05899-11 ◽

2011 ◽

Vol 193 (22) ◽

pp. 6276-6287 ◽

Cited By ~ 13

Author(s):

Boris R. Belitsky ◽

Abraham L. Sonenshein

Keyword(s):

Bacillus Subtilis ◽

Binding Sites ◽

Consensus Sequence ◽

Regulatory Region ◽

Binding Motif ◽

Downstream Site ◽

Upstream Site ◽

Binding Motifs ◽

Content Type ◽

A Minor

CodY is a global transcriptional regulator known to control expression of more than 100 genes and operons inBacillus subtilis. Some of the most strongly repressed targets of CodY, thenupNOPQ(formerly,yufNOPQ) genes, were found to encode a guanosine transporter. Using DNase I footprinting experiments, we identified two high-affinity CodY-binding sites in the regulatory region of thenupNgene. The two sites are located 50 bp upstream and 163 bp downstream of the transcription start site. The downstream site was responsible for 6- to 8-foldnupNrepression in the absence of the upstream site. When the upstream site was intact, however, only a minor contribution of the downstream site tonupNregulation could be detected under the conditions tested. Both sites contained 15-bp CodY-binding motifs with two mismatches each with respect to the consensus sequence, AATTTTCWGTTTTAA. However, the experimentally determined binding sites included additional sequences flanking the 15-bp CodY-binding motifs. An additional version of the 15-bp CodY-binding motif, with 5 mismatches with respect to the consensus but essential for efficient regulation by CodY, was found within the upstream site. The presence of multiple 15-bp motifs may be a common feature of CodY-binding sites.

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Structural characterization of a prolyl aminodipeptidase (PepX) from Lactobacillus helveticus

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19011774 ◽

2019 ◽

Vol 75 (10) ◽

pp. 625-633

Author(s):

Deanna Dahlke Ojennus ◽

Nicholas J. Bratt ◽

Kent L. Jones ◽

Douglas H. Juers

Keyword(s):

Calcium Ion ◽

Calcium Binding ◽

Consensus Sequence ◽

Lactobacillus Helveticus ◽

Binding Motif ◽

Molecular Replacement ◽

Food Allergens ◽

Peptide Bonds ◽

Starting Model

Prolyl aminodipeptidase (PepX) is an enzyme that hydrolyzes peptide bonds from the N-terminus of substrates when the penultimate amino-acid residue is a proline. Prolyl peptidases are of particular interest owing to their ability to hydrolyze food allergens that contain a high percentage of proline residues. PepX from Lactobacillus helveticus was cloned and expressed in Escherichia coli as an N-terminally His-tagged recombinant construct and was crystallized by hanging-drop vapor diffusion in a phosphate buffer using PEG 3350 as a precipitant. The structure was determined at 2.0 Å resolution by molecular replacement using the structure of PepX from Lactococcus lactis (PDB entry 1lns) as the starting model. Notable differences between the L. helveticus PepX structure and PDB entry 1lns include a cysteine instead of a phenylalanine at the substrate-binding site in the position which confers exopeptidase activity and the presence of a calcium ion coordinated by a calcium-binding motif with the consensus sequence DX(DN)XDG.

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Design of a companion bioinformatic tool to detect the emergence and geographical distribution of SARS-CoV-2 Spike protein genetic variants

Journal of Translational Medicine ◽

10.1186/s12967-020-02675-4 ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Alice Massacci ◽

Eleonora Sperandio ◽

Lorenzo D’Ambrosio ◽

Mariano Maffei ◽

Fabio Palombo ◽

...

Keyword(s):

Receptor Binding ◽

Consensus Sequence ◽

Cell Epitope ◽

Vaccine Design ◽

Binding Domain ◽

Spike Protein ◽

Antibody Activity ◽

Binding Motif ◽

Receptor Binding Domain ◽

The Impact

Abstract Background Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. Methods Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. Results Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. Conclusions This work provides a framework able to track down SARS-CoV-2 genomic variability.

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A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology inChlamydomonas reinhardtii

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904587116 ◽

2019 ◽

Vol 116 (37) ◽

pp. 18445-18454 ◽

Cited By ~ 14

Author(s):

Alan K. Itakura ◽

Kher Xing Chan ◽

Nicky Atkinson ◽

Leif Pallesen ◽

Lianyong Wang ◽

...

Keyword(s):

Surface Area ◽

Structure And Function ◽

Binding Motif ◽

Starch Granules ◽

Wild Type ◽

Bisphosphate Carboxylase ◽

Biophysical Mechanism ◽

Mechanism Function ◽

And Function

A phase-separated, liquid-like organelle called the pyrenoid mediates CO2fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model algaChlamydomonasthat has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant’s phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO2-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

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