scholarly journals The hns Gene of Escherichia coli Is Transcriptionally Down-Regulated by (p)ppGpp

2020 ◽  
Vol 8 (10) ◽  
pp. 1558
Author(s):  
Anna Brandi ◽  
Mara Giangrossi ◽  
Attilio Fabbretti ◽  
Maurizio Falconi
Keyword(s):  
Rna Polymerase ◽  
Transcriptional Repression ◽  
Mobility Shift ◽  
In Vitro System ◽  
Promoter Complex ◽  
The Stability ◽  
Electrophoretic Mobility Shift Assays ◽  
Fine Tune

Second messenger nucleotides, such as guanosine penta- or tetra-phosphate, commonly referred to as (p)ppGpp, are powerful signaling molecules, used by all bacteria to fine-tune cellular metabolism in response to nutrient availability. Indeed, under nutritional starvation, accumulation of (p)ppGpp reduces cell growth, inhibits stable RNAs synthesis, and selectively up- or down- regulates the expression of a large number of genes. Here, we show that the E. colihns promoter responds to intracellular level of (p)ppGpp. hns encodes the DNA binding protein H-NS, one of the major components of bacterial nucleoid. Currently, H-NS is viewed as a global regulator of transcription in an environment-dependent mode. Combining results from relA (ppGpp synthetase) and spoT (ppGpp synthetase/hydrolase) null mutants with those from an inducible plasmid encoded RelA system, we have found that hns expression is inversely correlated with the intracellular concentration of (p)ppGpp, particularly in exponential phase of growth. Furthermore, we have reproduced in an in vitro system the observed in vivo (p)ppGpp-mediated transcriptional repression of hns promoter. Electrophoretic mobility shift assays clearly demonstrated that this unusual nucleotide negatively affects the stability of RNA polymerase-hns promoter complex. Hence, these findings demonstrate that the hns promoter is subjected to an RNA polymerase-mediated down-regulation by increased intracellular levels of (p)ppGpp.

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.

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 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.

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 Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity

2004 ◽  
Vol 186 (18) ◽  
pp. 6254-6264 ◽  
Author(s):  
Peggy Freede ◽  
Sabine Brantl
Keyword(s):  
Binding Affinity ◽  
Copy Number ◽  
Dissociation Constants ◽  
Mobility Shift ◽  
Copy Numbers ◽  
Maximal Binding ◽  
Equilibrium Dissociation Constants ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5′-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 μM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3′ positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

scholarly journals The Sequence-specific DNA Binding of NF-κB Is Reversibly Regulated by the Automodification Reaction of Poly (ADP-ribose) Polymerase 1

2001 ◽  
Vol 276 (50) ◽  
pp. 47664-47670 ◽  
Author(s):  
Woo-Jin Chang ◽  
Rafael Alvarez-Gonzalez
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Specific Binding ◽  
High Specificity ◽  
Immunoblot Analysis ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays ◽  
Parp 1

Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-κB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-κB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+in vitro.Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-κB-p50 DNA binding was dependent on the presence of βNAD+. DNA binding by NF-κB-p50 was not efficient in the absence of βNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-κB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-κB-p50 with high specificity in the absence of βNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-κB-p50 to its DNA by inhibiting the specific protein·protein interactions between NF-κB-p50 and PARP-1. We also report the activation of NF-κB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-κB activationin vivoas well.

scholarly journals Binding of the RING Polycomb Proteins to Specific Target Genes in Complex with the grainyhead-Like Family of Developmental Transcription Factors

2002 ◽  
Vol 22 (6) ◽  
pp. 1936-1946 ◽  
Author(s):  
Annabel Tuckfield ◽  
David R. Clouston ◽  
Tomasz M. Wilanowski ◽  
Lin-Lin Zhao ◽  
John M. Cunningham ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Target Genes ◽  
Regulatory Elements ◽  
Polycomb Group ◽  
Mobility Shift ◽  
Polycomb Proteins ◽  
Complex Forms ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT The Polycomb group (PcG) of proteins represses homeotic gene expression through the assembly of multiprotein complexes on key regulatory elements. The mechanisms mediating complex assembly have remained enigmatic since most PcG proteins fail to bind DNA. We now demonstrate that the human PcG protein dinG interacts with CP2, a mammalian member of the grainyhead-like family of transcription factors, in vitro and in vivo. The functional consequence of this interaction is repression of CP2-dependent transcription. The CP2-dinG interaction is conserved in evolution with the Drosophila factor grainyhead binding to dring, the fly homologue of dinG. Electrophoretic mobility shift assays demonstrate that the grh-dring complex forms on regulatory elements of genes whose expression is repressed by grh but not on elements where grh plays an activator role. These observations reveal a novel mechanism by which PcG proteins may be anchored to specific regulatory elements in developmental genes.

scholarly journals The local transcriptional regulators SacR1 and SacR2 act as repressors of fructooligosaccharides metabolism in Lactobacillus plantarum

2020 ◽  
Author(s):  
Chen Chen ◽  
Linlin Wang ◽  
Haiyan Yu ◽  
huaixiang tian
Keyword(s):  
Lactobacillus Plantarum ◽  
Consensus Sequence ◽  
Protein A ◽  
Pcr Analysis ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Global And Local ◽  
Electrophoretic Mobility Shift Assays

Abstract Background: In Lactobacillus plantarum , fructooligosaccharides (FOS) metabolism is controlled by both global and local regulatory mechanisms. Although catabolite control protein A has been identified as a global regulator of FOS metabolism, the functions of local regulators remain unclear. This study aimed to elucidate the roles of two local regulators, SacR1 and SacR2, in the regulation of FOS metabolism in L. plantarum both in vitro and in vivo . Results: The inactivation of sacR1 and sacR2 affected the growth and production of metabolites for strains grown on FOS or glucose, respectively. A reverse transcription-quantitative PCR analysis of one wild-type and two mutant strains ( ΔsacR1 and ΔsacR2 ) of L. plantarum identified SacR1 and SacR2 as repressors of genes relevant to FOS metabolism in the absence of FOS, and these genes could be induced or derepressed by the addition of FOS. The analysis predicted four potential transcription factor binding sites (TFBSs) in the putative promoter regions of two FOS-related clusters. The binding of SacR1 and SacR2 to these TFBSs both in vitro and in vivo was verified using electrophoretic mobility shift assays and chromatin immunoprecipitation, respectively. A consensus sequence of WNNNNNAACGNNTTNNNNNW was deduced for the TFBSs of SacR1 and SacR2. Conclusion: Our results identified SacR1 and SacR2 as local repressors for FOS metabolism in L. plantarum . The regulation is achieved by the binding of SacR1 and SacR2 to TFBSs in the promoter regions of FOS-related clusters. The results provide new insights into the complex network regulating oligosaccharide metabolism by lactic acid bacteria .

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