scholarly journals The ArgP Protein Stimulates the Klebsiella pneumoniae gdhA Promoter in a Lysine-Sensitive Manner

2008 ◽  
Vol 190 (12) ◽  
pp. 4351-4359 ◽  
Author(s):  
Thomas J. Goss
Keyword(s):  
Klebsiella Pneumoniae ◽  
Upstream Region ◽  
Mobility Shift ◽  
Base Pairs ◽  
Dnase I Footprinting ◽  
Sensitive Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Knockout Mutation

ABSTRACT The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.

scholarly journals Interaction of the Gifsy-1 Xis Protein with the Gifsy-1 attP Sequence

2007 ◽  
Vol 189 (17) ◽  
pp. 6303-6311 ◽  
Author(s):  
Asa Flanigan ◽  
Jeffrey F. Gardner
Keyword(s):  
Dimethyl Sulfate ◽  
Direct Repeats ◽  
Mobility Shift ◽  
Base Pairs ◽  
Recombination Mechanism ◽  
Site Specific ◽  
Dnase I Footprinting ◽  
Pair Sequence ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Gifsy-1 phage integrates site specifically into the Salmonella chromosome via an integrase-mediated site-specific recombination mechanism. Initial genetic analysis suggests that Gifsy-1 integrase-mediated excision of the Gifsy-1 phage is influenced by proteins encoded by both the Gifsy-1 and the Gifsy-2 phages. Our studies show that the Gifsy-1 Xis protein regulates the directionality of integrase-mediated excision of the Gifsy-1 phage. Electrophoretic mobility shift assays, DNase I footprinting, dimethyl sulfate (DMS) interference assays, and DMS protection assays were used to identify a 31-base-pair sequence in the attP region to which the Gifsy-1 protein binds. The results suggest that this recombination directionality factor binds in vitro to three imperfect direct repeats, spaced 10 base pairs apart, in a sequential and cooperative manner in the absence of other phage-encoded proteins. Our studies suggest that, while the Gifsy-1 Xis does not require additional factors for specific and high-affinity binding, it may form a microfilament on DNA similar to that described for the phage lambda Xis protein.

Estrogen-inducible binding of a nuclear factor to the vitellogenin upstream region

1988 ◽  
Vol 8 (10) ◽  
pp. 4557-4560
Author(s):  
O Bakker ◽  
J N Philipsen ◽  
B C Hennis ◽  
G Ab
Keyword(s):  
Dimethyl Sulfate ◽  
Upstream Region ◽  
Nuclear Factor ◽  
Base Pairs ◽  
Exonuclease Iii ◽  
Factor I ◽  
Nuclear Factor I ◽  
Vitellogenin Gene

The estrogen-dependent binding of a protein to the upstream region of the chicken vitellogenin gene was detected by using in vivo dimethyl sulfate, genomic DNase I, and in vitro exonuclease III footprinting. The site is located between base pairs -848 and -824, and its sequence resembles that of the nuclear factor I binding site. The results suggest that a nuclear factor binding to this site is involved in the regulation of the vitellogenin gene.

scholarly journals SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

2007 ◽  
Vol 282 (46) ◽  
pp. 33326-33335 ◽  
Author(s):  
David Corbett ◽  
Hayley J. Bennett ◽  
Hamdia Askar ◽  
Jeffrey Green ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Regulation Of Transcription ◽  
Temperature Dependent ◽  
Mobility Shift ◽  
E Coli ◽  
Dnase I Footprinting ◽  
Nucleoprotein Complex ◽  
Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

scholarly journals Gata4 Regulates Testis Expression of Dmrt1

2004 ◽  
Vol 24 (1) ◽  
pp. 377-388 ◽  
Author(s):  
Ning Lei ◽  
Leslie L. Heckert
Keyword(s):  
Transient Transfection ◽  
Mobility Shift ◽  
Specific Expression ◽  
Dnase I Footprinting ◽  
Unknown Factor ◽  
Related Transcription Factor ◽  
Important Regulator ◽  
Functional Relevance ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The doublesex and mab-3 related transcription factor 1 (Dmrt1) is a putative transcriptional regulator that is expressed exclusively in the gonads and is required for postnatal testis differentiation. Here we describe the transcriptional mechanisms regulating testis-specific expression of the Dmrt1 gene. Transient-transfection analysis identified a region of the promoter between kb −3.2 and −2.8 that is important for Sertoli cell-specific expression. DNase I footprinting revealed four sites of DNA-protein interaction within this region, three of which were prominent in primary Sertoli cells. Analysis of these sites, using electrophoretic mobility shift assays, revealed that Gata4 and another unknown factor bound within these regions. Further transient-transfection assays of various mutant promoters established the functional relevance of the Gata4-response and unknown factor-response elements, while studies of Dmrt1 expression in 13.5 days postcoitum Fog2 null gonads supported the in vivo importance of Gata4's regulation. As a whole, these studies identify Gata4 as an important regulator in the Dmrt1 transcriptional machinery that is responsible for robust expression of Dmrt1 in the testis.

3,5-Diiodo-L-thyronine (T2) has selective thyromimetic effects in vivo and in vitro

1997 ◽  
Vol 19 (2) ◽  
pp. 137-147 ◽  
Author(s):  
SG Ball ◽  
J Sokolov ◽  
WW Chin
Keyword(s):  
Gh3 Cells ◽  
Mrna Levels ◽  
Thyroid Status ◽  
Hormone Activity ◽  
Mobility Shift ◽  
Nuclear Extracts ◽  
Electrophoretic Mobility Shift Assays ◽  
Gh3 Cell

Recent data have suggested that the iodothyronine, 3,5-diiodo-l-thyronine (T2), has selective thyromimetic activity. In vivo, T2 has been shown to suppress TSH levels at doses that do not produce significant peripheral manifestations of thyroid hormone activity. Furthermore, T2 has been shown to produce smaller increments in peripheral indices of thyroid status than does T3, when doses resulting in equivalent suppression of circulating TSH are compared. We have assessed the selective thyromimetic activity of T2 in vivo and in vitro, and performed in vitro studies to assess the potential molecular basis for these selective properties. T2 was 100-fold less potent than T3 in stimulating GH mRNA levels in GH3 cells. In contrast, the iodothyronines were almost equivalent in their ability to downregulate TRbeta2 mRNA levels in this cell line. Both 3,3'-diiodo-L-thyronine and thyronine exhibited no significant thyromimetic effects on either process. In vivo, doses of T2 and T3 that were equivalent in their induction of hepatic malic enzyme (ME) mRNA did not produce equivalent suppression of circulating TSH, with T2 being only 27% as effective as T3. T2 was up to 500-fold less potent than T3 in displacing [125I]-T3 from in vitro translated specific nuclear receptors (TRs) and GH3 cell nuclear extracts. Electrophoretic mobility shift assays, assessing the ability of T2 to produce dissociation of TRbeta1 homodimers from inverted palindrome T3 response elements, indicated that T2 was also 1000-fold less potent than T3 in this respect. These data confirm that T2 has significant thyromimetic activity, and that this activity is selective both in vivo and in vitro. However, there are no data to support a selective central effect, T2 being relatively more potent in stimulating hepatic ME mRNA than in suppression of TSH in vivo. The basis for this differential thyromimetic activity is not selective affinity of the different TR isoforms for T2, or divergent properties of T2 in competitive binding and functional assays in vitro.

scholarly journals Transcriptional Analysis of the grlRA Virulence Operon from Citrobacter rodentium

2010 ◽  
Vol 192 (14) ◽  
pp. 3722-3734 ◽  
Author(s):  
Marija Tauschek ◽  
Ji Yang ◽  
Dianna Hocking ◽  
Kristy Azzopardi ◽  
Aimee Tan ◽  
...  
Keyword(s):  
Core Promoter ◽  
Regulatory Protein ◽  
Transcriptional Analysis ◽  
Upstream Region ◽  
Citrobacter Rodentium ◽  
Dnase I Footprinting ◽  
Intestinal Pathogens ◽  
Enterocyte Effacement

ABSTRACT The locus for enterocyte effacement (LEE) is the virulence hallmark of the attaching-and-effacing (A/E) intestinal pathogens, namely, enteropathogenic Escherichia coli, enterohemorrhagic E. coli, and Citrobacter rodentium. The LEE carries more than 40 genes that are arranged in several operons, e.g., LEE1 to LEE5. Expression of the various transcriptional units is subject to xenogeneic silencing by the histone-like protein H-NS. The LEE1-encoded regulator, Ler, plays a key role in relieving this repression at several major LEE promoters, including LEE2 to LEE5. To achieve appropriate intracellular concentrations of Ler in different environments, A/E pathogens have evolved a sophisticated regulatory network to control ler expression. For example, the LEE-encoded GrlA and GrlR proteins work as activator and antiactivator, respectively, of ler transcription. Thus, control of the transcriptional activities of the LEE1 (ler) promoter and the grlRA operon determines the rate of transcription of all of the LEE-encoded virulence factors. To date, only a single promoter has been identified for the grlRA operon. In this study, we showed that the non-LEE-encoded AraC-like regulatory protein RegA of C. rodentium directly stimulates transcription of the grlRA promoter by binding to an upstream region in the presence of bicarbonate ions. In addition, in vivo and in vitro transcription assays revealed a σ70 promoter that is specifically responsible for transcription of grlA. Expression from this promoter was strongly repressed by H-NS and its paralog StpA but was activated by Ler. DNase I footprinting demonstrated that Ler binds to a region upstream of the grlA promoter, whereas H-NS interacts specifically with a region extending from the grlA core promoter into its coding sequence. Together, these findings provide new insights into the environmental regulation and differential expressions of the grlR and grlA genes of C. rodentium.

Regulation of the Bacillus subtilis mannitol utilization genes: promoter structure and transcriptional activation by the wild-type regulator (MtlR) and its mutants

Microbiology ◽  
2014 ◽  
Vol 160 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Kambiz Morabbi Heravi ◽  
Josef Altenbuchner
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Phosphotransferase System ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Proposed Model ◽  
Rna Polymerase Holoenzyme ◽  
Electrophoretic Mobility Shift Assays

Expression of mannitol utilization genes in Bacillus subtilis is directed by P mtlA , the promoter of the mtlAFD operon, and P mtlR , the promoter of the MtlR activator. MtlR contains phosphoenolpyruvate-dependent phosphotransferase system (PTS) regulation domains, called PRDs. The activity of PRD-containing MtlR is mainly regulated by the phosphorylation/dephosphorylation of its PRDII and EIIBGat-like domains. Replacing histidine 342 and cysteine 419 residues, which are the targets of phosphorylation in these two domains, by aspartate and alanine provided MtlR-H342D C419A, which permanently activates P mtlA in vivo. In the mtlR-H342D C419A mutant, P mtlA was active, even when the mtlAFD operon was deleted from the genome. The mtlR-H342D C419A allele was expressed in an Escherichia coli strain lacking enzyme I of the PTS. Electrophoretic mobility shift assays using purified MtlR-H342D C419A showed an interaction between the MtlR double-mutant and the Cy5-labelled P mtlA and P mtlR DNA fragments. These investigations indicate that the activated MtlR functions regardless of the presence of the mannitol-specific transporter (MtlA). This is in contrast to the proposed model in which the sequestration of MtlR by the MtlA transporter is necessary for the activity of MtlR. Additionally, DNase I footprinting, construction of P mtlA -P licB hybrid promoters, as well as increasing the distance between the MtlR operator and the −35 box of P mtlA revealed that the activated MtlR molecules and RNA polymerase holoenzyme likely form a class II type activation complex at P mtlA and P mtlR during transcription initiation.

scholarly journals E2A expression, nuclear localization, and in vivo formation of DNA- and non-DNA-binding species during B-cell development.

1993 ◽  
Vol 13 (12) ◽  
pp. 7321-7333 ◽  
Author(s):  
Y Jacobs ◽  
C Vierra ◽  
C Nelson
Keyword(s):  
B Cell ◽  
Electrophoretic Mobility ◽  
Cell Lines ◽  
Nuclear Localization ◽  
Cell Development ◽  
B Cell Development ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays

A monoclonal antibody (Yae) was characterized and shown to specifically recognize E2A proteins in vivo, including the E2A-Pbx1 fusion gene products, p77E2A-Pbx1 and p85E2A-Pbx1. E2A proteins of a predominant molecular mass of 72 kDa, which comigrated with in vitro-produced rat E12 and and rat E47, were detected in human pro-B, pre-B, mature B, and plasma cell lines. The Yae antibody detected an E2A-containing microE2 enhancer element-binding complex (BCF-1) in pre-B- and mature B-cell lines in electrophoretic mobility shift assays which displayed a migration rate similar to that of in vitro-produced rat E12 and rat E47. A new E2A-containing microE2-binding species (P-E2A) was identified in plasma cells by using electrophoretic mobility shift assays. E2A proteins were detected in pro-B cells but were unable to bind the microE2 site. These observations suggest that the microE2 site is the target of stage-specific E2A regulatory complexes during B-cell development. Immunostaining analyses demonstrated the predominant nuclear localization of E2A proteins. Finally, we have identified an E2A form, designated I-E2A, which is unable to bind DNA. Our observations demonstrate novel in vivo mechanisms for the regulation of transcription by E2A proteins during B-cell development.

scholarly journals MdMYB3 helps regulate anthocyanin accumulation in apple calli under moderately acidic conditions

2018 ◽  
Author(s):  
Yi-Cheng Wang ◽  
Jing-Jing Sun ◽  
Yan-Fen Qiu ◽  
Xiao-Jun Gong ◽  
Li Ma ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Myb Transcription Factor ◽  
Anthocyanin Content ◽  
Anthocyanin Accumulation ◽  
Mobility Shift ◽  
In The Wild ◽  
Acidic Conditions ◽  
Electrophoretic Mobility Shift Assays

AbstractAnthocyanins are the key factors controlling the coloration of plant tissues. However, the molecular mechanism underlying the effects of environmental pH on the synthesis of apple anthocyanins is unclear. In this study, we analyzed the anthocyanin contents of apple calli cultured in media at different pHs (5.5, 6.0, and 6.5). The highest anthocyanin content was observed at pH 6.0. Additionally, the moderately acidic conditions up-regulated the expression of MdMYB3 as well as specific anthocyanin biosynthesis structural genes (MdDFR and MdUFGT). Moreover, the anthocyanin content was higher in calli overexpressing MdMYB3 than in the wild-type controls at different pHs. Yeast one-hybrid assay results indicated that MdMYB3 binds to the MdDFR and MdUFGT promoters in vivo. An analysis of the MdDFR and MdUFGT promoters revealed multiple MYB-binding sites. Meanwhile, electrophoretic mobility shift assays confirmed that MdMYB3 binds to the MdDFR and MdUFGT promoters in vitro. Furthermore, GUS promoter activity assays suggested that the MdDFR and MdUFGT promoter activities are enhanced by acidic conditions, and the binding of MdMYB3 may further enhance activity. These results implied that an acid-induced apple MYB transcription factor (MdMYB3) promotes anthocyanin accumulation by up-regulating the expression of MdDFR and MdUFGT under moderately acidic conditions.

scholarly journals RNA Binding Properties of the Ty1 LTR-Retrotransposon Gag Protein

2021 ◽  
Vol 22 (16) ◽  
pp. 9103
Author(s):  
Julita Gumna ◽  
Angelika Andrzejewska-Romanowska ◽  
David J. Garfinkel ◽  
Katarzyna Pachulska-Wieczorek
Keyword(s):  
Rna Structure ◽  
Rna Binding ◽  
Rna Packaging ◽  
Packaging Signal ◽  
Mobility Shift ◽  
Gag Protein ◽  
Rna Dimerization ◽  
Electrophoretic Mobility Shift Assays

A universal feature of retroelement propagation is the formation of distinct nucleoprotein complexes mediated by the Gag capsid protein. The Ty1 retrotransposon Gag protein from Saccharomyces cerevisiae lacks sequence homology with retroviral Gag, but is functionally related. In addition to capsid assembly functions, Ty1 Gag promotes Ty1 RNA dimerization and cyclization and initiation of reverse transcription. Direct interactions between Gag and retrotransposon genomic RNA (gRNA) are needed for Ty1 replication, and mutations in the RNA-binding domain disrupt nucleation of retrosomes and assembly of functional virus-like particles (VLPs). Unlike retroviral Gag, the specificity of Ty1 Gag-RNA interactions remain poorly understood. Here we use microscale thermophoresis (MST) and electrophoretic mobility shift assays (EMSA) to analyze interactions of immature and mature Ty1 Gag with RNAs. The salt-dependent experiments showed that Ty1 Gag binds with high and similar affinity to different RNAs. However, we observed a preferential interaction between Ty1 Gag and Ty1 RNA containing a packaging signal (Psi) in RNA competition analyses. We also uncover a relationship between Ty1 RNA structure and Gag binding involving the pseudoknot present on Ty1 gRNA. In all likelihood, the differences in Gag binding affinity detected in vitro only partially explain selective Ty1 RNA packaging into VLPs in vivo.

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