Regulatory factors controlling transcription of Saccharomyces cerevisiae IXR1 by oxygen levels: a model of transcriptional adaptation from aerobiosis to hypoxia implicating ROX1 and IXR1 cross-regulation

2009 ◽  
Vol 425 (1) ◽  
pp. 235-243 ◽  
Author(s):  
Raquel  Castro-Prego ◽  
Mónica Lamas-Maceiras ◽  
Pilar Soengas ◽  
Isabel Carneiro ◽  
Isabel González-Siso ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Oxygen Availability ◽  
Mrna Levels ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Cross Links ◽  
Mobility Shift Assay ◽  
Transcriptional Adaptation

Ixr1p from Saccharomyces cerevisiae has been previously studied because it binds to DNA containing intrastrand cross-links formed by the anticancer drug cisplatin. Ixr1p is also a transcriptional regulator of anaerobic/hypoxic genes, such as SRP1/TIR1, which encodes a stress-response cell wall manoprotein, and COX5B, which encodes the Vb subunit of the mitochondrial complex cytochrome c oxidase. However, factors controlling IXR1 expression remained unexplored. In the present study we show that IXR1 mRNA levels are controlled by oxygen availability and increase during hypoxia. In aerobiosis, low levels of IXR1 expression are maintained by Rox1p repression through the general co-repressor complex Tup1–Ssn6. Ixr1p itself is necessary for full IXR1 expression under hypoxic conditions. Deletion analyses have identified the region in the IXR1 promoter responsible for this positive auto-control (nucleotides −557 to −376). EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays show that Ixr1p binds to the IXR1 promoter both in vitro and in vivo. Ixr1p is also required for hypoxic repression of ROX1 and binds to its promoter. UPC2 deletion has opposite effects on IXR1 and ROX1 transcription during hypoxia. Ixr1p is also necessary for resistance to oxidative stress generated by H2O2. IXR1 expression is moderately activated by H2O2 and this induction is Yap1p-dependent. A model of IXR1 regulation as a relay for sensing different signals related to change in oxygen availability is proposed. In this model, transcriptional adaptation from aerobiosis to hypoxia depends on ROX1 and IXR1 cross-regulation.

scholarly journals Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae.

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534 ◽  
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Mutational and in vitro protein-binding studies on centromere DNA from Saccharomyces cerevisiae

1987 ◽  
Vol 7 (12) ◽  
pp. 4522-4534
Author(s):  
R Ng ◽  
J Carbon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Binding ◽  
In Vitro Assays ◽  
Binding Studies ◽  
Mobility Shift ◽  
Sequence Element ◽  
Sequence Elements ◽  
Mobility Shift Assay

Centromeres on chromosomes in the yeast Saccharomyces cerevisiae contain approximately 140 base pairs (bp) of DNA. The functional centromere (CEN) region contains three important sequence elements (I, PuTCACPuTG; II, 78 to 86 bp of high-AT DNA; and III, a conserved 25-bp sequence with internal bilateral symmetry). Various point mutations or deletions in the element III region have a profound effect on CEN function in vivo, indicating that this DNA region is a key protein-binding site. This has been confirmed by the use of two in vitro assays to detect binding of yeast proteins to DNA fragments containing wild-type or mutationally altered CEN3 sequences. An exonuclease III protection assay was used to demonstrate specific binding of proteins to the element III region of CEN3. In addition, a gel DNA fragment mobility shift assay was used to characterize the binding reaction parameters. Sequence element III mutations that inactivate CEN function in vivo also prevent binding of proteins in the in vitro assays. The mobility shift assay indicates that double-stranded DNAs containing sequence element III efficiently bind proteins in the absence of sequence elements I and II, although the latter sequences are essential for optimal CEN function in vivo.

Transcriptional regulation mechanisms of hypoxia-induced neuroglobin gene expression

2012 ◽  
Vol 443 (1) ◽  
pp. 153-164 ◽  
Author(s):  
Ning Liu ◽  
Zhanyang Yu ◽  
Shuanglin Xiang ◽  
Song Zhao ◽  
Anna Tjärnlund-Wolf ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hypoxia Inducible Factor ◽  
Gene Promoter ◽  
Nuclear Factor Κb ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Hypoxic Conditions ◽  
Mobility Shift Assay

Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.

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 Transcript analysis reveals an extended regulon and the importance of protein–protein co-operativity for the Escherichia coli methionine repressor

2006 ◽  
Vol 396 (2) ◽  
pp. 227-234 ◽  
Author(s):  
Ferenc Marincs ◽  
Iain W. Manfield ◽  
Jonathan A. Stead ◽  
Kenneth J. Mcdowall ◽  
Peter G. Stockley
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Dna Arrays ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Metabolic Role ◽  
Mobility Shift Assay

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5′ promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein–protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein–protein and protein–DNA affinity.

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences

1989 ◽  
Vol 9 (11) ◽  
pp. 4706-4712
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Lacz Gene ◽  
Wild Type ◽  
Mobility Shift ◽  
Proline Utilization

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

scholarly journals MSY2 and MSY4 Bind a Conserved Sequence in the 3′ Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

2001 ◽  
Vol 21 (20) ◽  
pp. 7010-7019 ◽  
Author(s):  
Flaviano Giorgini ◽  
Holly G. Davies ◽  
Robert E. Braun
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Binding Activity ◽  
Mobility Shift ◽  
Conserved Sequence ◽  
Mobility Shift Assay ◽  
Protamine 1

ABSTRACT Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5′-UCCAUCA-3′, present in the 3′ untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of UACCACAUCCACU(subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.

scholarly journals Nucleotides Critical for the Interaction of the Streptococcus pyogenes Mga Virulence Regulator with Mga-Regulated Promoter Sequences

2012 ◽  
Vol 194 (18) ◽  
pp. 4904-4919 ◽  
Author(s):  
Lara L. Hause ◽  
Kevin S. McIver
Keyword(s):  
Binding Site ◽  
Streptococcus Pyogenes ◽  
Transcriptional Activation ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Content Type ◽  
Promoter Sequences ◽  
Mobility Shift Assay

ABSTRACTThe Mga regulator ofStreptococcus pyogenesdirectly activates the transcription of a core regulon that encodes virulence factors such as M protein (emm), C5a peptidase (scpA), and streptococcal inhibitor of complement (sic) by directly binding to a 45-bp binding site as determined by an electrophoretic mobility shift assay (EMSA) and DNase I protection. However, by comparing the nucleotide sequences of all established Mga binding sites, we found that they exhibit only 13.4% identity with no discernible symmetry. To determine the core nucleotides involved in functional Mga-DNA interactions, the M1T1 Pemm1binding site was altered and screened for nucleotides important for DNA bindingin vitroand for transcriptional activation using a plasmid-based luciferase reporterin vivo. Following this analysis, 34 nucleotides within the Pemm1binding site that had an effect on Mga binding, Mga-dependent transcriptional activation, or both were identified. Of these critical nucleotides, guanines and cytosines within the major groove were disproportionately identified clustered at the 5′ and 3′ ends of the binding site and with runs of nonessential adenines between the critical nucleotides. On the basis of these results, a Pemm1minimal binding site of 35 bp bound Mga at a level comparable to the level of binding of the larger 45-bp site. Comparison of Pemmwith directed mutagenesis performed in the M1T1 Mga-regulated PscpAand Psicpromoters, as well as methylation interference analysis of PscpA, establish that Mga binds to DNA in a promoter-specific manner.

scholarly journals The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

1989 ◽  
Vol 9 (11) ◽  
pp. 4706-4712 ◽  
Author(s):  
A H Siddiqui ◽  
M C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Complex Formation ◽  
Binding Activity ◽  
Lacz Gene ◽  
Wild Type ◽  
Mobility Shift ◽  
Proline Utilization

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.

scholarly journals GntR-like SCO3932 Protein Provides a Link between Actinomycete Integrative and Conjugative Elements and Secondary Metabolism

2021 ◽  
Vol 22 (21) ◽  
pp. 11867
Author(s):  
Krzysztof J. Pawlik ◽  
Mateusz Zelkowski ◽  
Mateusz Biernacki ◽  
Katarzyna Litwinska ◽  
Pawel Jaworski ◽  
...  
Keyword(s):  
Secondary Metabolism ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Neighboring Gene ◽  
Mobility Shift ◽  
Integrative And Conjugative Elements ◽  
Mobility Shift Assay ◽  
Metabolite Synthesis

Streptomyces bacteria produce a plethora of secondary metabolites including the majority of medically important antibiotics. The onset of secondary metabolism is correlated with morphological differentiation and controlled by a complex regulatory network involving numerous regulatory proteins. Control over these pathways at the molecular level has a medical and industrial importance. Here we describe a GntR-like DNA binding transcription factor SCO3932, encoded within an actinomycete integrative and conjugative element, which is involved in the secondary metabolite biosynthesis regulation. Affinity chromatography, electrophoresis mobility shift assay, footprinting and chromatin immunoprecipitation experiments revealed, both in vitro and in vivo, SCO3932 binding capability to its own promoter region shared with the neighboring gene SCO3933, as well as promoters of polyketide metabolite genes, such as cpkD, a coelimycin biosynthetic gene, and actII-orf4—an activator of actinorhodin biosynthesis. Increased activity of SCO3932 target promoters, as a result of SCO3932 overproduction, indicates an activatory role of this protein in Streptomyces coelicolor A3(2) metabolite synthesis pathways.

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