Comparison of in vitro and in vivo binding sites competition of Bacillus thuringiensis Cry1 proteins in two important maize pests

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

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Progastrin1–80stimulates growth of intestinal epithelial cells in vitro via high-affinity binding sites

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00351.2002 ◽

2003 ◽

Vol 284 (2) ◽

pp. G328-G339 ◽

Cited By ~ 41

Author(s):

P. Singh ◽

X. Lu ◽

S. Cobb ◽

B. T. Miller ◽

N. Tarasova ◽

...

Keyword(s):

Epithelial Cells ◽

Binding Sites ◽

Intestinal Epithelial Cells ◽

Full Length ◽

Intestinal Epithelial ◽

Growth Effects ◽

High Affinity ◽

Significant Difference

Proliferation and carcinogenesis of the large intestinal epithelial cells (IEC) cells is significantly increased in transgenic mice that overexpress the precursor progastrin (PG) peptide. It is not known if the in vivo growth effects of PG on IEC cells are mediated directly or indirectly. Full-length recombinant human PG (rhPG1–80) was generated to examine possible direct effects of PG on IEC cells. Surprisingly, rhPG (0.1–1.0 nM) was more effective than the completely processed gastrin 17 (G17) peptide as a growth factor. Even though IEC cells did not express CCK1and CCK2receptors (-R), fluorescently labeled G17 and Gly-extended G17 (G-Gly) were specifically bound to the cells, suggesting the presence of binding proteins other than CCK1-R and CCK2-R on IEC cells. High-affinity ( Kd= 0.5–1.0 nM) binding sites for125I-rhPG were discovered on IEC cells that demonstrated relative binding affinity for gastrin-like peptides in the order PG ≥ COOH-terminally extended G17 ≥ G-Gly > G17 > *CCK-8 (* significant difference; P< 0.05). In conclusion, our studies demonstrate for the first time direct growth effects of the full-length precursor peptide on IEC cells in vitro that are apparently mediated by the high-affinity PG binding sites that were discovered on these cells.

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On in vitro and in vivo binding of tobacco mosaic virus to cytoplasmic membranes

Biochemie und Physiologie der Pflanzen ◽

10.1016/s0015-3796(84)80090-6 ◽

1984 ◽

Vol 179 (6) ◽

pp. 507-516 ◽

Cited By ~ 4

Author(s):

Barbara Pustowoit ◽

Wladimir Pustowoit ◽

Gottfried Schuster

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

In Vivo Binding ◽

Cytoplasmic Membranes

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Temperature-dependence of the DnaA–DNA interaction and its effect on the autoregulation of dnaA expression

Biochemical Journal ◽

10.1042/bj20120876 ◽

2012 ◽

Vol 449 (2) ◽

pp. 333-341 ◽

Cited By ~ 6

Author(s):

Chiara Saggioro ◽

Anne Olliver ◽

Bianca Sclavi

Keyword(s):

Temperature Dependence ◽

Binding Sites ◽

Dna Interaction ◽

Bacterial Dna ◽

Dnaa Protein ◽

High Affinity ◽

Key Factor

The DnaA protein is a key factor for the regulation of the timing and synchrony of initiation of bacterial DNA replication. The transcription of the dnaA gene in Escherichia coli is regulated by two promoters, dnaAP1 and dnaAP2. The region between these two promoters contains several DnaA-binding sites that have been shown to play an important role in the negative auto-regulation of dnaA expression. The results obtained in the present study using an in vitro and in vivo quantitative analysis of the effect of mutations to the high-affinity DnaA sites reveal an additional effect of positive autoregulation. We investigated the role of transcription autoregulation in the change of dnaA expression as a function of temperature. While negative auto-regulation is lost at dnaAP1, the effects of both positive and negative autoregulation are maintained at the dnaAP2 promoter upon lowering the growth temperature. These observations can be explained by the results obtained in vitro showing a difference in the temperature-dependence of DnaA–ATP binding to its high- and low-affinity sites, resulting in a decrease in DnaA–ATP oligomerization at lower temperatures. The results of the present study underline the importance of the role for autoregulation of gene expression in the cellular adaptation to different growth temperatures.

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SSMART: Sequence-structure motif identification for RNA-binding proteins

10.1101/287953 ◽

2018 ◽

Cited By ~ 2

Author(s):

Alina Munteanu ◽

Neelanjan Mukherjee ◽

Uwe Ohler

Keyword(s):

Binding Sites ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Sequence Motif ◽

Primary Sequence ◽

Sequence Structure ◽

Rna Targets

AbstractMotivationRNA-binding proteins (RBPs) regulate every aspect of RNA metabolism and function. There are hundreds of RBPs encoded in the eukaryotic genomes, and each recognize its RNA targets through a specific mixture of RNA sequence and structure properties. For most RBPs, however, only a primary sequence motif has been determined, while the structure of the binding sites is uncharacterized.ResultsWe developed SSMART, an RNA motif finder that simultaneously models the primary sequence and the structural properties of the RNA targets sites. The sequence-structure motifs are represented as consensus strings over a degenerate alphabet, extending the IUPAC codes for nucleotides to account for secondary structure preferences. Evaluation on synthetic data showed that SSMART is able to recover both sequence and structure motifs implanted into 3‘UTR-like sequences, for various degrees of structured/unstructured binding sites. In addition, we successfully used SSMART on high-throughput in vivo and in vitro data, showing that we not only recover the known sequence motif, but also gain insight into the structural preferences of the RBP.AvailabilitySSMART is freely available at https://ohlerlab.mdc-berlin.de/software/SSMART_137/[email protected]

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The closely related Drosophila sry beta and sry delta zinc finger proteins show differential embryonic expression and distinct patterns of binding sites on polytene chromosomes

Development ◽

10.1242/dev.110.1.141 ◽

1990 ◽

Vol 110 (1) ◽

pp. 141-149 ◽

Cited By ~ 1

Author(s):

F. Payre ◽

S. Noselli ◽

V. Lefrere ◽

A. Vincent

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Polytene Chromosomes ◽

Duplication Event ◽

Amino Acid Sequences ◽

Evolutionary Divergence ◽

Coding Region ◽

Protein Coding

Serendipity (sry) beta (beta) and delta (delta) are two finger protein genes resulting from a duplication event. Comparison of their respective protein products shows interspersed blocks of conserved and divergent amino-acid sequences. The most extensively conserved region corresponds to the predicted DNA-binding domain which includes 6 contiguous fingers; no significant sequence conservation is found upstream and downstream of the protein-coding region. We have analysed the evolutionary divergence of the sry beta and delta proteins on two separate levels, their embryonic pattern of expression and their DNA-binding properties in vitro and in vivo. By using specific antibodies and transformant lines containing beta-galactosidase fusion genes, we show that the sry beta and sry delta proteins are maternally inherited and present in embryonic nuclei at the onset of zygotic transcription, suggesting that they are transcription factors involved in this process. Zygotic synthesis of the sry beta protein starts during nuclear division cycles 12–13, prior to cellularisation of the blastoderm, while the zygotic sry delta protein is not detectable before germ band extension (stage 10 embryos). Contrary to sry delta, the zygotic sry beta protein constitutes only a minor fraction of the total embryonic protein. The sry beta and delta proteins made in E. coli bind to DNA, with partly overlapping specificities. Their in vivo patterns of binding to DNA, visualised by immunostaining polytene chromosomes, differ both in the number and position of their binding sites. Thus changes in expression pattern and DNA-binding specificity have contributed to the evolution of the sry beta and delta genes.

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Partial inhibition by cyclosporin A of the swelling of liver mitochondria in vivo and in vitro induced by sub-micromolar [Ca2+], but not by butyrate. Evidence for two distinct swelling mechanisms

Biochemical Journal ◽

10.1042/bj2680147 ◽

1990 ◽

Vol 268 (1) ◽

pp. 147-152 ◽

Cited By ~ 66

Author(s):

A M Davidson ◽

A P Halestrap

Keyword(s):

Light Scattering ◽

Cyclosporin A ◽

Binding Sites ◽

Mitochondrial Protein ◽

Liver Mitochondria ◽

Partial Inhibition ◽

Mitochondrial Volume ◽

Bongkrekic Acid

1. The effects of cyclosporin A on the increase in matrix PPi and consequent swelling of energized liver mitochondria incubated with 1 mM-butyrate, 30 microM-bongkrekic acid or 0.1-35 microM-Ca2+ [Halestrap (1989) Biochim. Biophys. Acta 973, 355-382] were studied. 2. Cyclosporin (1 microM) had no significant effect on the swelling induced by butyrate, bongkrekic acid or Ca2+ at concentrations of less than 0.3 microM. 3. At higher [Ca2+] (greater than 0.3 microM), swelling became progressively inhibited by cyclosporin, although the increase in matrix PPi was slightly greater in the presence than in the absence of cyclosporin. 4. Titration with cyclosporin indicated that there are 128 pmol of relevant cyclosporin-binding sites per mg of mitochondrial protein, with a Ki of about 5 nM. 5. The decrease in light-scattering by hepatocytes induced by butyrate [Davidson & Halestrap (1988) Biochem. J. 254, 379-384] was unaffected by cyclosporin, whereas that induced by vasopressin was inhibited by 20-30% without a significant change in cellular PPi content. 6. It is suggested that there are two mechanisms for the increase in mitochondrial volume induced by Ca2+: a PPi-mediated mechanism that is insensitive to cyclosporin and an additional Ca2(+)-mediated effect that is inhibited by cyclosporin. The nature of these pathways and their inter-relationship is discussed in the following paper [Halestrap & Davidson (1990) Biochem. J. 268, 153-160].

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Structural characterization of amorphous calcium carbonate-binding protein: an insight into the mechanism of amorphous calcium carbonate formation

Biochemical Journal ◽

10.1042/bj20130285 ◽

2013 ◽

Vol 453 (2) ◽

pp. 179-186 ◽

Cited By ~ 14

Author(s):

Jingtan Su ◽

Xiao Liang ◽

Qiang Zhou ◽

Guiyou Zhang ◽

Hongzhong Wang ◽

...

Keyword(s):

Calcium Carbonate ◽

Binding Sites ◽

Binding Protein ◽

Homology Modelling ◽

Size Exclusion ◽

Structural Basis ◽

Amorphous Calcium Carbonate ◽

Carbonate Formation

ACC (amorphous calcium carbonate) plays an important role in biomineralization process for its function as a precursor for calcium carbonate biominerals. However, it is unclear how biomacromolecules regulate the formation of ACC precursor in vivo. In the present study, we used biochemical experiments coupled with bioinformatics approaches to explore the mechanisms of ACC formation controlled by ACCBP (ACC-binding protein). Size-exclusion chromatography, chemical cross-linking experiments and negative staining electron microscopy reveal that ACCBP is a decamer composed of two adjacent pentamers. Sequence analyses and fluorescence quenching results indicate that ACCBP contains two Ca2+-binding sites. The results of in vitro crystallization experiments suggest that one Ca2+-binding site is critical for ACC formation and the other site affects the ACC induction efficiency. Homology modelling demonstrates that the Ca2+-binding sites of pentameric ACCBP are arranged in a 5-fold symmetry, which is the structural basis for ACC formation. To the best of our knowledge, this is the first report on the structural basis for protein-induced ACC formation and it will significantly improve our understanding of the amorphous precursor pathway.

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