Integration host factor interactions with Neisseria gene sequences: correlation between predicted binding sites and in vitro binding of Neisseria -derived IHF protein

2002 ◽  
Vol 16 (2) ◽  
pp. 153-158 ◽  
Author(s):  
S.A. Hill ◽  
D.S. Samuels ◽  
C. Nielsen ◽  
S.W. Knight ◽  
F. Pagotto ◽  
...  
Keyword(s):  
Binding Sites ◽  
Integration Host Factor ◽  
Host Factor ◽  
Gene Sequences ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Factor Interactions ◽  
Ihf Protein

scholarly journals In Vitro Selection of Integration Host Factor Binding Sites

1999 ◽  
Vol 181 (10) ◽  
pp. 3246-3255 ◽  
Author(s):  
Steven D. Goodman ◽  
Nerissa J. Velten ◽  
Qian Gao ◽  
Scott Robinson ◽  
Anca M. Segall
Keyword(s):  
Binding Sites ◽  
In Vitro Selection ◽  
Structural Characteristics ◽  
Integration Host Factor ◽  
Host Factor ◽  
Binding Domain ◽  
Physical Constraints ◽  
Individual Sequences ◽  
And Function

ABSTRACT Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3′ domain is conserved, the 5′ domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5′ domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5′ binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5′ binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.

scholarly journals TraY and Integration Host Factor oriT Binding Sites and F Conjugal Transfer: Sequence Variations, but Not Altered Spacing, Are Tolerated

2007 ◽  
Vol 189 (10) ◽  
pp. 3813-3823 ◽  
Author(s):  
Sarah L. Williams ◽  
Joel F. Schildbach
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Integration Host Factor ◽  
Host Factor ◽  
Conjugative Plasmid ◽  
Sequence Specificity ◽  
Single Stranded Region ◽  
Plasmid Mobilization ◽  
Dna Strand

ABSTRACT Bacterial conjugation is the process by which a single strand of a conjugative plasmid is transferred from donor to recipient. For F plasmid, TraI, a relaxase or nickase, binds a single plasmid DNA strand at its specific origin of transfer (oriT) binding site, sbi, and cleaves at a site called nic. In vitro studies suggest TraI is recruited to sbi by its accessory proteins, TraY and integration host factor (IHF). TraY and IHF bind conserved oriT sites sbyA and ihfA, respectively, and bend DNA. The resulting conformational changes may propagate to nic, generating the single-stranded region that TraI can bind. Previous deletion studies performed by others showed transfer efficiency of a plasmid containing F oriT decreased progressively as increasingly longer segments, ultimately containing both sbyA and ihfA, were deleted. Here we describe our efforts to more precisely define the role of sbyA and ihfA by examining the effects of multiple base substitutions at sbyA and ihfA on binding and plasmid mobilization. While we observed significant decreases in in vitro DNA-binding affinities, we saw little effect on plasmid mobilization even when sbyA and ihfA variants were combined. In contrast, when half or full helical turns were inserted between the relaxosome protein-binding sites, mobilization was dramatically reduced, in some cases below the detectable limit of the assay. These results are consistent with TraY and IHF recognizing sbyA and ihfA with limited sequence specificity and with relaxosome proteins requiring proper spacing and orientation with respect to each other.

scholarly journals Synthesis and in vitro binding of N,N-dialkyl-2-phenylindol-3-yl-glyoxylamides for the peripheral benzodiazepine binding sites

2006 ◽  
Vol 14 (11) ◽  
pp. 3938-3946 ◽  
Author(s):  
Taryn P. Homes ◽  
Filomena Mattner ◽  
Paul A. Keller ◽  
Andrew Katsifis
Keyword(s):  
Binding Sites ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Benzodiazepine Binding ◽  
Benzodiazepine Binding Sites

scholarly journals In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated ς54-Dependent Po Promoter

2001 ◽  
Vol 183 (9) ◽  
pp. 2842-2851 ◽  
Author(s):  
Chun Chau Sze ◽  
Andrew D. Laurie ◽  
Victoria Shingler
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Binding Sites ◽  
Integration Host Factor ◽  
Host Factor ◽  
Physical Interaction ◽  
Rich Region ◽  
Polymerase Binding

ABSTRACT Transcription from the Pseudomonas CF600-derived ς54-dependent promoter Po is controlled by the aromatic-responsive activator DmpR. Here we examine the mechanism(s) by which integration host factor (IHF) stimulates DmpR-activated transcriptional output of the Po promoter both in vivo and in vitro. In vivo, the Po promoter exhibits characteristics that typify many ς54-dependent promoters, namely, a phasing-dependent tolerance with respect to the distance from the regulator binding sites to the distally located RNA polymerase binding site, and a strong dependence on IHF for optimal promoter output. IHF is shown to affect transcription via structural repercussions mediated through binding to a single DNA signature located between the regulator and RNA polymerase binding sites. In vitro, using DNA templates that lack the regulator binding sites and thus bypass a role of IHF in facilitating physical interaction between the regulator and the transcriptional apparatus, IHF still mediates a DNA binding-dependent stimulation of Po transcription. This stimulatory effect is shown to be independent of previously described mechanisms for the effects of IHF at ς54 promoters such as aiding binding of the regulator or recruitment of ς54-RNA polymerase via UP element-like DNA. The effect of IHF could be traced to promotion and/or stabilization of open complexes within the nucleoprotein complex that may involve an A+T-rich region of the IHF binding site and promoter-upstream DNA. Mechanistic implications are discussed in the context of a model in which IHF binding results in transduction of DNA instability from an A+T-rich region to the melt region of the promoter.

Muscarinic regulation of phospholipase A2 and iodide fluxes in FRTL-5 thyroid cells

1991 ◽  
Vol 125 (2) ◽  
pp. 192-200 ◽  
Author(s):  
Maria Di Girolamo ◽  
Daniela D'Arcangelo ◽  
Cinzia Bizzarri ◽  
Daniela Corda
Keyword(s):  
Arachidonic Acid ◽  
Adenylyl Cyclase ◽  
Muscarinic Receptor ◽  
Binding Sites ◽  
Signal Pathways ◽  
Thyroid Cells ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Stimulation Of

Abstract. FRTL-5 thyroid cells express a muscarinic receptor which inhibits the phospholipase C activity in a pirenzepine-insensitive manner. We here report that the cholinergic agonist carbachol decreases in these cells the steady-state iodide content, an effect correlated with the iodination of thyroglobulin and with thyroid hormone formation. Several signal pathways may be involved in this phenomenon since carbachol in addition to inhibiting phospholipase C, increased the arachidonic acid release and modified the adenylyl cyclase activity. In FRTL-5 cells, arachidonic acid is released via the direct stimulation of phospholipase A2 by a pirenzepine-sensitive muscarinic receptor coupled to a GTP binding protein sensitive to pertussis toxin. Regarding adenylyl cyclase, carbachol potentiated the thyrotropin-induced stimulation of the enzyme, whereas it did not affect the basal levels of cAMP. In vitro binding studies revealed the presence of two muscarinic binding sites. To summarize, the analysis of signal pathways and of in vitro binding sites indicates a complex muscarinic regulation of thyroid function, which includes the modulation of iodide fluxes.

Identification of potential binding sites for the FHA domain of human Chk2 by in vitro binding studies

2003 ◽  
Vol 311 (4) ◽  
pp. 803-808 ◽  
Author(s):  
Dongyan Qin ◽  
Hyun Lee ◽  
Chunhua Yuan ◽  
Yong Ju ◽  
Ming-Daw Tsai
Keyword(s):  
Binding Sites ◽  
Binding Studies ◽  
Fha Domain ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Potential Binding

Species differences in mGluR5 binding sites in mammalian central nervous system determined using in vitro binding with [18F]F-PEB

2007 ◽  
Vol 34 (8) ◽  
pp. 1009-1017 ◽  
Author(s):  
Shil Patel ◽  
Terence G. Hamill ◽  
Brett Connolly ◽  
Elaine Jagoda ◽  
Wenping Li ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Binding Sites ◽  
Species Differences ◽  
In Vitro Binding ◽  
Mammalian Central Nervous System ◽  
Vitro Binding

scholarly journals Potential of the Bacillus thuringiensis Toxin Reservoir for the Control of Lobesia botrana (Lepidoptera: Tortricidae), a Major Pest of Grape Plants

2006 ◽  
Vol 73 (1) ◽  
pp. 337-340 ◽  
Author(s):  
Iñigo Ruiz de Escudero ◽  
Anna Estela ◽  
Baltasar Escriche ◽  
Primitivo Caballero
Keyword(s):  
Bacillus Thuringiensis ◽  
Binding Sites ◽  
Lobesia Botrana ◽  
Active Components ◽  
Cry Proteins ◽  
In Vitro Binding ◽  
First Instar ◽  
Vitro Binding ◽  
Major Pest

ABSTRACT The potential of Bacillus thuringiensis Cry proteins to control the grape pest Lobesia botrana was explored by testing first-instar larvae with Cry proteins belonging to the Cry1, Cry2, and Cry9 groups selected for their documented activities against Lepidoptera. Cry9Ca, a toxin from B. thuringiensis, was the protein most toxic to L. botrana larvae, followed in decreasing order by Cry2Ab, Cry1Ab, Cry2Aa, and Cry1Ia7, with 50% lethal concentration values of 0.09, 0.1, 1.4, 3.2, and 8.5 μg/ml of diet, respectively. In contrast, Cry1Fa and Cry1JA were not active at the assayed concentration (100 μg/ml). In vitro binding and competition experiments showed that none of the toxins tested (Cry1Ia, Cry2Aa, Cry2Ab, and Cry9C) shared binding sites with Cry1Ab. We conclude that either Cry1Ia or Cry9C could be used in combination with Cry1Ab to control this pest, either as the active components of B. thuringiensis sprays or expressed together in transgenic plants.

Discovery of New AKT1 Inhibitors by Combination of In silico Structure Based Virtual Screening Approaches and Biological Evaluations

2019 ◽  
Author(s):  
Filip Fratev ◽  
Denisse A. Gutierrez ◽  
Renato J. Aguilera ◽  
suman sirimulla
Keyword(s):  
Virtual Screening ◽  
Success Rate ◽  
Protein Interactions ◽  
In Silico ◽  
Biological Data ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
Screening Protocol ◽  
Screening Approaches

AKT1 is emerging as a useful target for treating cancer. Herein, we discovered a new set of ligands that inhibit the AKT1, as shown by in vitro binding and cell line studies, using a newly designed virtual screening protocol that combines structure-based pharmacophore and docking screens. Taking together with the biological data, the combination of structure based pharamcophore and docking methods demonstrated reasonable success rate in identifying new inhibitors (60-70%) proving the success of aforementioned approach. A detail analysis of the ligand-protein interactions was performed explaining observed activities.<br>

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