Specific binding of the Xanthomonas campestris pv. vesicatoria AraC-type transcriptional activator HrpX to plant-inducible promoter boxes.;Koebnik R, Krüger A, Thieme F, Urban A, Bonas U;Journal of bacteriology 2006 Nov;
188(21):7652-60
[16936021]
Genome-wide consensus search for PIP boxes was performed. Results were then validated by RT-PCR.
To determine whether HrpX binds the PIP box, we used a magnetic DNA affinity enrichment approach.
A 300-bp DNA fragment harboring the two PIP boxes (14 bp from each other) of the divergently transcribed hrpB and hrpC operons was amplified by PCR. The purified DNA fragments were immobilized on streptavidin-coated magnetic beads, which were then incubated with a crude protein extract from X. campestris pv. vesicatoria containing HrpX-His. After washing and elution of bound proteins from the beads, eluted proteins were analyzed by SDS-PAGE and Western blot analysis using His tag-specific antibodies.
To confirm the sequence specificity of HrpX binding, DNA fragments mutated in the PIP box were used. For this experiment, we constructed three mutants with substitutions in the hrpD PIP box.
Regulated genes for each binding site are displayed below. Gene regulation diagrams
show binding sites, positively-regulated genes,
negatively-regulated genes,
both positively and negatively regulated
genes, genes with unspecified type of regulation.
For each indvidual site, experimental techniques used to determine the site are also given.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
Quantitative Reverse-Transcription PCR is a modification of PCR in which RNA is first reverse transcribed into cDNA and this is amplified measuring the product (qPCR) in real time. It therefore allows one to analyze transcription by directly measuring the product (RNA) of a gene's transcription. If the gene is transcribed more, the starting product for PCR is larger and the corresponding volume of amplification is also larger.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
In DNA affinity purification, DNA from promoter regions thought to be bound by the protein is labelled (e.g. biotinylated) and bound to beads (or some type of matrix). The protein is then left to interact with DNA and eventually eluted. Bound protein will remain attached to beads. This can be detected through gel electophoresis. The protein can then be sequenced by mass-spectrometry. The techinque can be used to demonstrate binding of a purified protein, or to purify the binding protein from crude extract or a mix of proteins.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
In DNA affinity purification, DNA from promoter regions thought to be bound by the protein is labelled (e.g. biotinylated) and bound to beads (or some type of matrix). The protein is then left to interact with DNA and eventually eluted. Bound protein will remain attached to beads. This can be detected through gel electophoresis. The protein can then be sequenced by mass-spectrometry. The techinque can be used to demonstrate binding of a purified protein, or to purify the binding protein from crude extract or a mix of proteins.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
In DNA affinity purification, DNA from promoter regions thought to be bound by the protein is labelled (e.g. biotinylated) and bound to beads (or some type of matrix). The protein is then left to interact with DNA and eventually eluted. Bound protein will remain attached to beads. This can be detected through gel electophoresis. The protein can then be sequenced by mass-spectrometry. The techinque can be used to demonstrate binding of a purified protein, or to purify the binding protein from crude extract or a mix of proteins.
Target-specific mutation, as opposed to non-specific mutation.
In the context of TF-binding sites, site-directed mutagenesis is typically used to establish/confirm the specific sequence and location of a site, often in tandem with EMSA.
Different positions of a putative binding site are mutated to non-consensus (or random) bases and binding to the mutated site is evaluated through EMSA or other means. Often implemented only in conserved motif positions or serially through all positions of a site.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.
In DNA affinity purification, DNA from promoter regions thought to be bound by the protein is labelled (e.g. biotinylated) and bound to beads (or some type of matrix). The protein is then left to interact with DNA and eventually eluted. Bound protein will remain attached to beads. This can be detected through gel electophoresis. The protein can then be sequenced by mass-spectrometry. The techinque can be used to demonstrate binding of a purified protein, or to purify the binding protein from crude extract or a mix of proteins.
This is a weak form of in-silico search, in which the consensus sequence for the motif is compared to genomic positions and the number of mismatches (between candidate site and consensus) is used as a measure of site-quality.