Characterization of Specific Nucleotide Substitutions in DtxR-Specific Operators of Corynebacterium diphtheriae That Dramatically Affect DtxR Binding, Operator Function, and Promoter Strength

ABSTRACT The diphtheria toxin repressor (DtxR) of Corynebacterium diphtheriae uses Fe2+ as a corepressor. Holo-DtxR inhibits transcription from the iron-regulated promoters (IRPs) designated IRP1 through IRP5 as well as from the promoters for thetox and hmuO genes. DtxR binds to 19-bp operators with the consensus sequence 5′-TTAGGTTAGCCTAACCTAA-3′, a perfect 9-bp palindrome interrupted by a single C · G base pair. Among the seven known DtxR-specific operators, IRP3 exhibits the weakest binding to DtxR. The message (sense) strand of the IRP3 operator (5′-TTAGGTGAGACGCACCCAT-3′ [nonconsensus nucleotides underlined]) overlaps by 2 nucleotides at its 5′ end with the putative −10 sequence of the IRP3 promoter. The underlined C at position +7 from the center of the IRP3 operator [C(+7)] is unique, because T is conserved at that position in other DtxR-specific operators. The present study examined the effects of nucleotide substitutions at position +7 or −7 in the IRP3 operator. In gel mobility shift assays, only the change of C(+7) to the consensus nucleotide T caused a dramatic increase in the binding of DtxR, whereas other nucleotide substitutions for C(+7) or replacements for A(−7) had only small positive or negative effects on DtxR binding. All substitutions for C(+7) or A(−7) except for A(−7)C dramatically decreased IRP3 promoter strength. In contrast, the A(−7)C variant caused increased promoter strength at the cost of nearly eliminating repressibility by DtxR. The message (sense) strand of the IRP1 operator (5′-TTAGGTTAGCCAAACCTTT-3′) includes the −35 region of the IRP3 promoter. A T(+7)C variant of the IRP1 operator was also constructed, and it was shown to exhibit decreased binding to DtxR, decreased repressibility by DtxR, and increased promoter strength. The nucleotides at positions +7 and −7 in DtxR-specific operators are therefore important determinants of DtxR binding and repressibility of transcription by DtxR, and they also have significant effects on promoter activity for IRP3 and IRP1.

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Characterization of BreR Interaction with the Bile Response PromotersbreABandbreRin Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.02008-12 ◽

2012 ◽

Vol 195 (2) ◽

pp. 307-317 ◽

Cited By ~ 6

Author(s):

Francisca A. Cerda-Maira ◽

Gabriela Kovacikova ◽

Brooke A. Jude ◽

Karen Skorupski ◽

Ronald K. Taylor

Keyword(s):

Vibrio Cholerae ◽

Transcriptional Repression ◽

Consensus Sequence ◽

Site Directed Mutagenesis ◽

Mobility Shift ◽

Content Type ◽

Proximal Site ◽

Bile Resistance ◽

Polymerase Binding ◽

Gel Mobility Shift

ABSTRACTTheVibrio choleraeBreR protein is a transcriptional repressor of thebreABefflux system operon, which encodes proteins involved in bile resistance. In a previous study (F. A. Cerda-Maira, C. S. Ringelberg, and R. K. Taylor, J. Bacteriol.190:7441–7452, 2008), we used gel mobility shift assays to determine that BreR binds at two independent binding sites at thebreABpromoter and a single site at its own promoter. Here it is shown, by DNase I footprinting and site-directed mutagenesis, that BreR is able to bind at a distal and a proximal site in thebreABpromoter. However, only one of these sites, the proximal 29-bp site, is necessary for BreR-mediated transcriptional repression ofbreABexpression. In addition, it was determined that BreR represses its own expression by recognizing a 28-bp site at thebreRpromoter. These sites comprise regions of dyad symmetry within which residues critical for BreR function could be identified. The BreR consensus sequence AANGTANAC-N6-GTNTACNTT overlaps the −35 region at both promoters, implying that the repression of gene expression is achieved by interfering with RNA polymerase binding at these promoters.

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Binding of Escherichia coli integration host factor (IHF) to the origin segment of p15A plasmid.

Acta Biochimica Polonica ◽

10.18388/abp.1995_4675 ◽

1995 ◽

Vol 42 (1) ◽

pp. 103-108

Author(s):

E Hiszczyńska-Sawicka ◽

J Kur

Keyword(s):

Escherichia Coli ◽

Consensus Sequence ◽

Integration Host Factor ◽

Host Factor ◽

Mobility Shift ◽

Consensus Sequences ◽

Report Data ◽

Functional Dna ◽

Gel Mobility Shift ◽

Dna Binding And Bending

The integration host factor (IHF) is a sequence-specific, histone-like, multi-functional DNA-binding and -bending protein of Escherichia coli. Characterization and functional analysis of this protein has been carried out mainly in bacteriophage lambda and other mobile genetic elements. In this paper we report data concerning the binding of IHF protein to the plasmid orip15A region. IHF binds to the single site of the DNA fragment containing the orip15A, as shown by the gel mobility shift assays and footprinting experiment. On the basis of the ihf consensus sequences published, we have been able to identify one sequence of putative ihf site into the orip15A sequence with two mismatches in relation to the consensus sequence of Kur et al., 1989, Gene 81, 1-15. One ihf binding site was also found in the oriColE1 region sequence with three mismatches in relation to this consensus sequence.

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A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1470-1475.1990 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1470-1475

Author(s):

A Yanagida ◽

K Sogawa ◽

K I Yasumoto ◽

Y Fujii-Kuriyama

Keyword(s):

Regulatory Element ◽

Consensus Sequence ◽

Inducible Expression ◽

Recognition Sequence ◽

Mobility Shift ◽

Enhancer Activity ◽

Cis Acting ◽

Tata Sequence ◽

Gel Mobility Shift ◽

High Level

A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.

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A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene.

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1470 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1470-1475 ◽

Cited By ~ 63

Author(s):

A Yanagida ◽

K Sogawa ◽

K I Yasumoto ◽

Y Fujii-Kuriyama

Keyword(s):

Regulatory Element ◽

Consensus Sequence ◽

Inducible Expression ◽

Recognition Sequence ◽

Mobility Shift ◽

Enhancer Activity ◽

Cis Acting ◽

Tata Sequence ◽

Gel Mobility Shift ◽

High Level

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Cross-Regulation of the Bacillus subtilis glnRA and tnrA Genes Provides Evidence for DNA Binding Site Discrimination by GlnR and TnrA

Journal of Bacteriology ◽

10.1128/jb.188.7.2578-2585.2006 ◽

2006 ◽

Vol 188 (7) ◽

pp. 2578-2585 ◽

Cited By ~ 32

Author(s):

Jill M. Zalieckas ◽

Lewis V. Wray ◽

Susan H. Fisher

Keyword(s):

Bacillus Subtilis ◽

Dna Binding ◽

Promoter Region ◽

Nitrogen Availability ◽

Mutational Analysis ◽

Consensus Sequence ◽

Amino Acid Sequences ◽

Mobility Shift ◽

Binding Domains ◽

Gel Mobility Shift

ABSTRACT Two Bacillus subtilis transcriptional factors, TnrA and GlnR, regulate gene expression in response to changes in nitrogen availability. These two proteins have similar amino acid sequences in their DNA binding domains and bind to DNA sites (GlnR/TnrA sites) that have the same consensus sequence. Expression of the tnrA gene was found to be activated by TnrA and repressed by GlnR. Mutational analysis demonstrated that a GlnR/TnrA site which lies immediately upstream of the −35 region of the tnrA promoter is required for regulation of tnrA expression by both GlnR and TnrA. Expression of the glnRA operon, which contains two GlnR/TnrA binding sites (glnRAo1 and glnRAo2) in its promoter region, is repressed by both GlnR and TnrA. The glnRAo2 site, which overlaps the −35 region of the glnRA promoter, was shown to be required for regulation by both GlnR and TnrA, while the glnRAo1 site which lies upstream of the −35 promoter region is only involved in GlnR-mediated regulation. Examination of TnrA binding to tnrA and glnRA promoter DNA in gel mobility shift experiments showed that TnrA bound with an equilibrium dissociation binding constant of 55 nM to the GlnR/TnrA site in the tnrA promoter region, while the affinities of TnrA for the two GlnR/TnrA sites in the glnRA promoter region were greater than 3 μM. These results demonstrate that GlnR and TnrA cross-regulate each other's expression and that there are differences in their DNA-binding specificities.

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Transcription from the P2 promoter of the growth hormone receptor gene involves members of the Sp transcription factor family

Biochemical Journal ◽

10.1042/bj3440867 ◽

1999 ◽

Vol 344 (3) ◽

pp. 867-872 ◽

Cited By ~ 5

Author(s):

Timothy E. ADAMS

Keyword(s):

Growth Hormone ◽

Transcription Factors ◽

Binding Sites ◽

Hormone Receptor ◽

Growth Hormone Receptor ◽

Consensus Sequence ◽

Mobility Shift ◽

Promoter Construct ◽

P2 Promoter ◽

Gel Mobility Shift

The P2 promoter of the gene for growth hormone receptor is developmentally regulated and is differentially active in a number of tissues. Little is known about the identity of the transcription factors that participate to effect this pattern of transcription. Deletion analysis and transient transfection were used to localize a previously identified cis-acting element within the sheep P2 promoter to between positions -99 and -87. Gel mobility-shift assays with nuclear extracts from Chinese hamster ovary (CHO-K1) fibroblasts revealed that this sequence encompasses an atypical binding site for both Sp1 and two isoforms of Sp3. A gel mobility-shift scan of promoter sequences between -88 and +21 indicated the existence of three other binding sites for Sp1 and Sp3. One of these, designated site II and found by using a probe spanning -74 to -54, corresponds to a classical GC box consensus sequence. Site III (-63 to -41) and site IV (-27 to -5) harbour atypical Sp1/Sp3-binding sequences. Site-directed mutagenesis of site II or site IV decreased promoter activity by approx. 40%, whereas a promoter construct incorporating both mutations exhibited negligible (approx. 1%) activity. Co-transfection of expression plasmids encoding either Sp1 or Sp3 significantly transactivated reporter gene activity from a P2 promoter construct carrying all four Sp1/Sp3-binding sites (8-fold compared with 7.1-fold induction respectively). Sp1 is known to interact with a variety of other transcription factors to regulate the transcription of a number of differentially expressed genes. The identification of four binding sites for Sp1 and Sp3 within the P2 promoter of the gene for growth hormone receptor might point to other factors that interact to regulate the activity of this promoter in different tissues during foetal and post-natal development.

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The Bacillus subtilis DinR Binding Site: Redefinition of the Consensus Sequence

Journal of Bacteriology ◽

10.1128/jb.180.8.2201-2211.1998 ◽

1998 ◽

Vol 180 (8) ◽

pp. 2201-2211 ◽

Cited By ~ 65

Author(s):

Kevin W. Winterling ◽

David Chafin ◽

Jeffery J. Hayes ◽

Ji Sun ◽

Arthur S. Levine ◽

...

Keyword(s):

Bacillus Subtilis ◽

Binding Site ◽

Mutational Analysis ◽

Consensus Sequence ◽

Nucleotide Substitutions ◽

Mobility Shift ◽

Radical Cleavage ◽

Computational Analyses ◽

Dyad Symmetry ◽

Electrophoretic Mobility Shift Assays

ABSTRACT Recently, the DinR protein was established as the cellular repressor of the SOS response in the bacterium Bacillus subtilis. It is believed that DinR functions as the repressor by binding to a consensus sequence located in the promoter region of each SOS gene. The binding site for DinR is believed to be synonymous with the formerly identified Cheo box, a region of 12 bp displaying dyad symmetry (GAAC-N4-GTTC). Electrophoretic mobility shift assays revealed that highly purified DinR does bind to such sites located upstream of the dinA, dinB,dinC, and dinR genes. Furthermore, detailed mutational analysis of the B. subtilis recA operator indicates that some nucleotides are more important than others for maintaining efficient DinR binding. For example, nucleotide substitutions immediately 5′ and 3′ of the Cheo box as well as those in the N4 region appear to affect DinR binding. This data, combined with computational analyses of potential binding sites in other gram-positive organisms, yields a new consensus (DinR box) of 5′-CGAACRNRYGTTYC-3′. DNA footprint analysis of the B. subtilis dinR and recA DinR boxes revealed that the DinR box is centrally located within a DNA region of 31 bp that is protected from hydroxyl radical cleavage in the presence of DinR. Furthermore, while DinR is predominantly monomeric in solution, it apparently binds to the DinR box in a dimeric state.

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Snapshot blotting: The transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to EM grids

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124215 ◽

1992 ◽

Vol 50 (1) ◽

pp. 762-763

Author(s):

Stephen D. Jett

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Nucleic Acid Binding ◽

Mobility Shift ◽

Carbon Coated ◽

Nucleoprotein Complexes ◽

Mobility Shift Assay ◽

Protein Nucleic Acid ◽

Gel Mobility Shift ◽

Nucleic Acid Interactions

The electrophoresis gel mobility shift assay is a popular method for the study of protein-nucleic acid interactions. The binding of proteins to DNA is characterized by a reduction in the electrophoretic mobility of the nucleic acid. Binding affinity, stoichiometry, and kinetics can be obtained from such assays; however, it is often desirable to image the various species in the gel bands using TEM. Present methods for isolation of nucleoproteins from gel bands are inefficient and often destroy the native structure of the complexes. We have developed a technique, called “snapshot blotting,” by which nucleic acids and nucleoprotein complexes in electrophoresis gels can be electrophoretically transferred directly onto carbon-coated grids for TEM imaging.

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Erfolg der Jungwaldpflege im Schweizer Mittelland? Analyse und Folgerungen (Essay)

Schweizerische Zeitschrift fur Forstwesen ◽

10.3188/szf.2013.0262 ◽

2013 ◽

Vol 164 (9) ◽

pp. 262-270 ◽

Cited By ~ 5

Author(s):

Peter Ammann

Keyword(s):

Stand Development ◽

Negative Effects ◽

Tree Species Composition ◽

Natural Processes ◽

Course Of Action ◽

Tree Thinning ◽

Swiss Plateau ◽

Self Differentiation ◽

The Cost ◽

Young Growth

Is young growth tending successful in the Swiss Plateau region? Analysis and implications (essay) The effect of the cost-intensive young growth tending used up to the present in the region of the Swiss Plateau is analysed using different approaches. It is evident that young growth tending is not only ineffective with respect to diameter growth but even hinders stand development. Negative effects on quality from young growth tending are also recognised. This is often due to premature interventions in the natural processes of self-differentiation and subsequent systematic errors in the thinning. Furthermore, the effect of tending measures on the tree species composition is often overestimated because in the first 10 to 20 years of stand development, it is primarily the rejuvenation strategy and the site which are decisive. As an alternative course of action, tending concepts are proposed which rely on biological rationalisation and future tree thinning, to achieve future trees which are as vigorous as possible. These are not only more effective, but are also significantly less expensive.

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