scholarly journals NDD1, a High-Dosage Suppressor ofcdc28-1N, Is Essential for Expression of a Subset of Late-S-Phase-Specific Genes in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (5) ◽  
pp. 3312-3327 ◽  
Author(s):  
Chong Jin Loy ◽  
David Lydall ◽  
Uttam Surana
Keyword(s):  
Binding Sites ◽  
Nuclear Division ◽  
Protein Complexes ◽  
Essential Gene ◽  
Promoter Sequence ◽  
S Phase ◽  
Restrictive Temperature ◽  
Mobility Shift ◽  
Dna Fragment ◽  
Gel Mobility Shift

ABSTRACT cdc28-1N mutants progress through the G1and S phases normally at the restrictive temperature but fail to undergo nuclear division. We have isolated a gene, NDD1, which at a high dosage suppresses the nuclear-division defect ofcdc28-1N. NDD1 (nuclear division defective) is an essential gene. Its expression during the cell cycle is tightly regulated such that NDD1 RNA is most abundant during the S phase. Cells lacking the NDD1 gene arrest with an elongated bud, a short mitotic spindle, 2N DNA content, and an undivided nucleus, suggesting that its function is required for some aspect of nuclear division. We show that overexpression of Ndd1 results in the upregulation of bothCLB1 and CLB2 transcription, suggesting that the suppression of cdc28-1N by NDD1 may be due to an accumulation of these cyclins. Overproduction of Ndd1 also enhances the expression of SWI5, whose transcription, like that of CLB1 and CLB2, is activated in the late S phase. Ndd1 is essential for the expression of CLB1,CLB2, and SWI5, since none of these genes are transcribed in its absence. Both CLB2 expression and its upregulation by NDD1 are mediated by a 240-bp promoter sequence that contains four MCM1-binding sites. However, Ndd1 does not appear to be a component of any of the protein complexes assembled on this DNA fragment, as indicated by gel mobility shift assays. Instead, overexpression of NDD1 prevents the formation of one of the complexes whose appearance correlates with the termination of CLB2 expression in G1. The inability of GAL1 promoter-driven CLB2 to suppress the lethality of NDD1 null mutant suggests that, in addition to CLB1 and CLB2, NDD1may also be required for the transcription of other genes whose functions are necessary for G2/M transition.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Identification of cis Elements Directing Termination of Yeast Nonpolyadenylated snoRNA Transcripts

2004 ◽  
Vol 24 (14) ◽  
pp. 6241-6252 ◽  
Author(s):  
Kristina L. Carroll ◽  
Dennis A. Pradhan ◽  
Josh A. Granek ◽  
Neil D. Clarke ◽  
Jeffry L. Corden
Keyword(s):  
Rna Polymerase Ii ◽  
Binding Sites ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Large Degree ◽  
Sequence Motifs ◽  
Mobility Shift ◽  
Nascent Transcript ◽  
Pol Ii ◽  
Gel Mobility Shift

ABSTRACT RNA polymerase II (Pol II) termination is triggered by sequences present in the nascent transcript. Termination of pre-mRNA transcription is coupled to recognition of cis-acting sequences that direct cleavage and polyadenylation of the pre-mRNA. Termination of nonpolyadenylated [non-poly(A)] Pol II transcripts in Saccharomyces cerevisiae requires the RNA-binding proteins Nrd1 and Nab3. We have used a mutational strategy to characterize non-poly(A) termination elements downstream of the SNR13 and SNR47 snoRNA genes. This approach detected two common RNA sequence motifs, GUA[AG] and UCUU. The first motif corresponds to the known Nrd1-binding site, which we have verified here by gel mobility shift assays. We also show that Nab3 protein binds specifically to RNA containing the UCUU motif. Taken together, our data suggest that Nrd1 and Nab3 binding sites play a significant role in defining non-poly(A) terminators. As is the case with poly(A) terminators, there is no strong consensus for non-poly(A) terminators, and the arrangement of Nrd1p and Nab3p binding sites varies considerably. In addition, the organization of these sequences is not strongly conserved among even closely related yeasts. This indicates a large degree of genetic variability. Despite this variability, we were able to use a computational model to show that the binding sites for Nrd1 and Nab3 can identify genes for which transcription termination is mediated by these proteins.

scholarly journals Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314 ◽  
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Identification of the DNA Binding Sites of PerA, the Transcriptional Activator of the bfp and per Operons in Enteropathogenic Escherichia coli

2003 ◽  
Vol 185 (9) ◽  
pp. 2835-2847 ◽  
Author(s):  
J. Antonio Ibarra ◽  
Miryam I. Villalba ◽  
José Luis Puente
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Promoter Sequence ◽  
Transcriptional Regulators ◽  
Mobility Shift ◽  
Dna Motifs ◽  
Dnase I Footprinting ◽  
Dna Binding Sites ◽  
Important Virulence Factor ◽  
Similar Affinity

ABSTRACT The bundle-forming pilus (BFP) is an important virulence factor for enteropathogenic Escherichia coli (EPEC). Genes involved in its biogenesis and regulation are tightly regulated by PerA (BfpT), a member of the AraC/XylS family of transcriptional regulators. The aim of this work was to purify PerA and determine its association with bfpA and perA (bfpT) regulatory regions by electrophoretic mobility shift and DNase I footprinting assays. PerA was purified as a maltose-binding protein (MBP) fusion, which was capable of complementing bfpA expression and which was able to restore the localized adherence phenotype of an EPEC perA mutant strain. Upstream of bfpA and perA, MBP-PerA recognized with similar affinity asymmetric nucleotide sequences in which a 29-bp-long AT-rich consensus motif was identified. These DNA motifs share 66% identity and were previously shown, by deletion analysis, to be involved in the PerA-dependent expression of both genes. Interestingly, in perA, this motif spans the sequence between positions −75 and −47, approximately one helix turn upstream of the −35 promoter sequence, while in bfpA, it spans the sequence between positions −83 and −55, approximately two helix turns upstream from the promoter. An additional PerA binding site was identified at the 5′ end of the bfpA structural gene, which was not required for its activation. Experiments with LexA-PerA fusions suggested that PerA acts as a monomer to activate the transcription of both perA and bfpA, in contrast to what has been documented for other members of this family of transcriptional regulators.

Molecular characterization of the CmbR activator-binding site in the metC–cysK promoter region in Lactococcus lactis

Microbiology ◽  
2005 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Natasa Golic ◽  
Martijn Schliekelmann ◽  
María Fernández ◽  
Michiel Kleerebezem ◽  
Richard van Kranenburg
Keyword(s):  
Binding Site ◽  
Lactococcus Lactis ◽  
Promoter Region ◽  
Direct Repeat ◽  
Inverted Repeats ◽  
Sulphur Metabolism ◽  
Mobility Shift ◽  
Dna Fragment ◽  
Gel Mobility Shift

The metC–cysK operon involved in sulphur metabolism in Lactococcus lactis is positively regulated by the LysR-type protein CmbR. Transcription from the metC promoter is activated when concentrations of methionine and cysteine in the growth medium are low. The metC promoter region contains two direct and three inverted repeats. Deletion analysis indicated that direct repeat 2 (DR2) is required for activation of the metC promoter by CmbR. Gel mobility shift assays confirmed that CmbR binds to a 407 bp DNA fragment containing the metC promoter. This binding was stimulated by O-acetyl-l-serine. Competition experiments with deletion variants of the metC promoter showed that CmbR binding only occurred with fragments containing an intact DR2, confirming that DR2 is the CmbR binding site within the metC promoter.

scholarly journals Members of the GATA Family of Transcription Factors Bind to the U3 Region of Cas-Br-E and Graffi Retroviruses and Transactivate Their Expression

1998 ◽  
Vol 72 (7) ◽  
pp. 5579-5588 ◽  
Author(s):  
Corinne Barat ◽  
Eric Rassart
Keyword(s):  
Transcription Factors ◽  
Binding Sites ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Nuclear Extracts ◽  
Gel Mobility Shift ◽  
Leukemia Viruses ◽  
Immature Cells ◽  
Granulocytic Leukemia ◽  
Gata Family

ABSTRACT Cas-Br-E and Graffi are two murine viruses that induce myeloid leukemia in mice: while Cas-Br-E induces mostly non-T, non-B leukemia composed of very immature cells, Graffi causes exclusively a granulocytic leukemia (E. Rassart, J. Houde, C. Denicourt, M. Ru, C. Barat, E. Edouard, L. Poliquin, and D. Bergeron, Curr. Top. Microbiol. Immunol. 211:201–210, 1995). In an attempt to understand the basis of the myeloid specificity of these two retroviruses, we used DNase I footprinting analysis and gel mobility shift assays to identify a number of protein binding sites within the Cas-Br-E and Graffi U3 regions. Two protected regions include potential GATA binding sites. Methylation interference analysis with different hematopoietic nuclear extracts showed the importance of the G residues in these GATA sites, and supershift assays clearly identified the binding factors as GATA-1, GATA-2, and GATA-3. Transient assays with long terminal repeat (LTR)-chloramphenicol acetyltransferase constructs showed that these three GATA family members are indeed able to transactivate Cas-Br-E and Graffi LTRs. Thus, the availability and relative abundance of the various members of the GATA family of transcription factors in a given cell type could influence the transcriptional tissue specificity of murine leukemia viruses and hence their disease specificity.

Activation of the NF-κB Pathway by Inflammatory Stimuli in Human Neutrophils

Blood ◽  
1997 ◽  
Vol 89 (9) ◽  
pp. 3421-3433 ◽  
Author(s):  
Patrick P. McDonald ◽  
Anette Bald ◽  
Marco A. Cassatella
Keyword(s):  
Cell Activation ◽  
Protein Complexes ◽  
Binding Activity ◽  
Human Neutrophils ◽  
Resting Cells ◽  
Mobility Shift ◽  
Dna Binding Activity ◽  
Cytokines And Chemokines ◽  
Inflammatory Stimuli ◽  
Gel Mobility Shift

Abstract Activated neutrophils have the ability to upregulate the expression of many genes, in particular those encoding cytokines and chemokines, and to subsequently release the corresponding proteins. Although little is known to date concerning the regulation of gene transcription in neutrophils, it is noteworthy that many of these genes depend on the activation of transcription factors, such as NF-κB, for inducible expression. We therefore investigated whether NF-κB/Rel proteins are expressed in human neutrophils, as well as their fate on cell activation. We now report that dimers consisting of p50 NFκB1, p65 RelA, and/or c-Rel are present in neutrophils and that the greater part of these protein complexes is physically associated with cytoplasmic IκB-α in resting cells. Following neutrophil stimulation with proinflammatory agonists (such as lipopolysaccharide [LPS], tumor necrosis factor-α [TNF-α], and fMet-Leu-Phe) that induce the production of cytokines and chemokines in these cells, NF-κB/Rel proteins translocated to nuclear fractions, resulting in a transient induction of NF-κB DNA binding activity, as determined in gel mobility shift assays. The onset of both processes was found to be closely paralleled by, and dependent on, IκB-α degradation. Proinflammatory neutrophil stimuli also promoted the accumulation of IκB-α mRNA transcripts, resulting in the reexpression of the IκB-α protein. To our knowledge, this constitutes the first indication that NF-κB activation may underlie the action of proinflammatory stimuli towards human neutrophil gene expression and, as such, adds a new facet to our understanding of neutrophil biology.

scholarly journals Characterization of cis-Acting Sites Controlling Arginine Deiminase Gene Expression in Streptococcus gordonii

2006 ◽  
Vol 188 (3) ◽  
pp. 941-949 ◽  
Author(s):  
Lin Zeng ◽  
Yiqian Dong ◽  
Robert A. Burne
Keyword(s):  
Binding Sites ◽  
Promoter Region ◽  
Sequence Similarity ◽  
Acid Tolerance ◽  
Arginine Deiminase ◽  
Streptococcus Gordonii ◽  
Oral Diseases ◽  
Mobility Shift ◽  
Gel Mobility Shift

ABSTRACT The arginine deiminase system (ADS) is responsible for the production of ornithine, CO2, ammonia, and ATP from arginine. The ADS of the oral bacterium Streptococcus gordonii plays major roles in physiologic homeostasis, acid tolerance, and oral biofilm ecology. To further our understanding of the transcriptional regulation of the ADS (arc) operon, the binding of the ArcR transcriptional activator, which governs expression of the ADS in response to arginine, was investigated by DNase I protection and gel mobility shift assays. An ArcR binding sequence was found that was 27 bp in length and had little sequence similarity to binding sites of other arginine metabolism regulators. The presence of arginine at physiologically relevant concentrations enhanced the binding of ArcR to its target. Using cat fusions, various deletion and substitution mutations within the putative ArcR footprint were shown to cause dramatic reductions in expression from the arcA promoter in vivo, confirming that the 27-bp sequence is required for optimal expression and induction of the ADS by arginine. Mutation of two putative catabolite response elements (CREs) within the arc promoter region showed that both CREs contribute to catabolite repression. A thorough understanding of the regulation of the ADS in S. gordonii and related organisms is needed to develop ways to exploit arginine catabolism for the control of oral diseases. Identification of the ArcR and CcpA binding sites lays the foundation for a more complete understanding of the complex interactions of multiple regulatory proteins with elements in the arc promoter region.

scholarly journals Activation of mouse Pi-class glutathione S-transferase gene by Nrf2(NF-E2-related factor 2) and androgen

2002 ◽  
Vol 364 (2) ◽  
pp. 563-570 ◽  
Author(s):  
Hiromi IKEDA ◽  
Mohamed S. SERRIA ◽  
Ikuko KAKIZAKI ◽  
Ichiro HATAYAMA ◽  
Kimihiko SATOH ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Receptor Binding ◽  
Binding Sites ◽  
Related Factor ◽  
Glutathione S Transferase ◽  
Mobility Shift ◽  
Enhancer Activity ◽  
Drug Induction ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

The Pi-class glutathione S-transferases (GSTs) play pivotal roles in the detoxification of xenobiotics, carcinogenesis and drug resistance. The mechanisms of regulation of these genes during drug induction and carcinogenesis are yet to be elucidated. Recently, Nrf2 (NF-E2-related factor 2; a bZip-type transcription factor) knockout mice were shown to display impaired induction of Pi-class GST genes by drugs. It is known that the mouse Pi-class GST gene GST-P1 is expressed predominantly in the male liver, and is regulated by androgen. To determine whether Nrf2 and the androgen receptor regulate GST-P1 directly, we analysed the molecular mechanism of activation of this gene by these factors. The promoter of the GST-P1 gene was activated markedly by Nrf2 in transient transfection analyses. Gel mobility shift assay and footprinting analyses revealed three Nrf2 binding sites: one at the proximal and two at distal elements, located at positions −59, −915 and −937 from the cap site. The fifth intron of the GST-P1 gene contains the androgen-responsive region. Multiple androgen receptor binding sites are clustered within a 500bp region of this intron. The whole fragment contains a minimum of seven androgen receptor binding sites, which collectively display strong androgen-dependent enhancer activity. However, on division into small fragments containing two or three elements each, individual enhancer activities were dramatically decreased. This suggests that multiple elements work synergistically as a strong androgen-responsive enhancer. Our findings indicate that Nrf2 and the androgen receptor directly bind to and activate the mouse GST-P1 gene.

Identification of the DNA-binding site of the Rgg-like regulator LasX within the lactocin S promoter region

Microbiology ◽  
2005 ◽  
Vol 151 (3) ◽  
pp. 813-823 ◽  
Author(s):  
Elizabeth L. Andersen Rawlinson ◽  
Ingolf F. Nes ◽  
Morten Skaugen
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Promoter Region ◽  
Direct Repeats ◽  
Mobility Shift ◽  
Dna Binding Site ◽  
Intergenic Dna ◽  
Dna Fragment ◽  
Derivatives Of

LasX regulates the transcription of the divergent operons lasXY and lasA–W, which specify the production of lactocin S in Lactobacillus sakei L45. Using histidine-tagged LasX, and a DNA fragment containing the complete intergenic lasA–lasX region, electrophoresis mobility-shift (EMSA) analyses were employed to demonstrate that LasX binds to the lasA–lasX intergenic DNA. Two direct heptanucleotide motifs directly upstream of P lasA–W , and a third imperfect copy of this motif, overlapping the −10 element of P lasA–W , were identified as possible LasX-binding sites. To assess the role of the direct repeats in the binding of LasX to the intergenic lasA–lasX region, binding experiments were performed using DNA probes with different combinations of the repeats, and with arbitrarily chosen repeat substitutions. The result of these experiments demonstrated that only the middle repeat was required for the binding of LasX to the las-promoter region. This observation correlated with the results of subsequent reporter-gene analyses, thereby weakening the hypothesis of the involvement of the direct repeats in LasX-mediated transcription regulation. By analysing the ability of LasX to bind successively shortened derivatives of the original intergenic fragment, a tentative 19 bp minimum LasX-binding site was identified.

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