2-D electrophoresis begins with 1-D electrophoresis but then separates the molecules by a second property in a direction 90 degrees from the first. In 1-D electrophoresis, proteins (or other molecules) are separated in one dimension, so that all the proteins/molecules will lie along a lane but that the molecules are spread out across a 2-D gel. The two dimensions that proteins are separated into using this technique can be isoelectric point, protein complex mass in the native state, and protein mass.
This technique is most commonly used when trying to establish a proteome for an organism, but can be used to demonstrate changes in expression, in the change in size/concentration of the spot on the gel that corresponds to the target protein.
A quantifiable phenotypic trait (e.g. glucose intake) is assessed in a quantitative manner after induction of the regulatory mechanism and used as a natural reporter..
The observable phenotypic trait should be described in the experimental notes.
Alkaline phosphatase reporter assay
Very similar in spirit to the standard beta-galactosidase reporter, in this assay genes coding for a protein of interest are fused with the phoA gene encoding alkaline phosphatase. The result is often a hybrid protein with alkaline phosphatase activity. PhoA becomes active only when it has been transported across the cellular membrane into the periplasmic space, allowing to trace activity of secreted proteins.
Beta-gal reporter assay
Reporter assay using the beta-galactosidase (lacZ) gene.
The lacZ gene is typically fused to the promoter of interest. Differential regulation of the promoter mediated by the TF is assessed by induction of the system and evaluation of lacZ expression. Bacteria expressing lacZ appear blue when grown on a X-gal medium.
The assay is often performed using a plasmid borne construction on a lacZ(def) strain.
A reporter assay based on the fusion of a promoter of interest with the cat gene, which produces chloramphenicol acetyltransferase, capable of being acetylated by acetyl CoA. The amount of acetylated chloramphenicol is directly proportional to the amount of CAT enzyme present.
The principle of ChIP-chip is simple. The first step is to cross-link the protein-DNA complex. This is done using a fixating agent, such as formaldehyde. The cross-linking can later be reversed with heat. Cross-linking kills the cell, giving a snapshot of the bound TF at a given time. The cell is then lysed, the DNA sheared by sonication and the chromatin (TF-DNA complexes) is pulled down using an antibody (i.e. immunoprecipitated). If an antibody for the TF is available, then it is used; otherwise, the TF is tagged with an epitope targeted by commercially available antibodies (the latter option is cheaper, but runs the risk of altering the TF's functionality). Cross-linking is then reversed to free the bound DNA, which is then amplified, labeled with a fluorophore and dumped onto a DNA-array. The scanned array reveals the genomic regions bound by the TF. The resolution is around ~500 bp as a result of the sonication step.
A variation of ChIP-seq involving the addition of an exonuclease that cleaves the ChIPed DNA that is not strictly protected by the bound protein, resulting in improved resolution of the binding site during the later sequencing step.
ChIP-chip (and to a lesser degree ChIP-Seq) results are often validated with ChIP-PCR, in which a PCR with specific primers is performed on the pulled-down DNA. As in the case of RNASeq, there are many variations of these main techniques.
ChIP-Seq is equivalent to ChIP-chip down to the last step. In ChIP-Seq, immunoprecipiated DNA fragments are prepared for sequencing and funneled into a massively parallel sequencer that produces short reads. Even though the sonication step is the same as in ChIP-chip, ChIP-Seq will generate multiple short-reads within any given 500 bp region, thereby pinning down the location of TFBS to within 50-100 bp. A similar result can be obtained with ChIP-chip using high-density tiling-arrays. The downside of ChIP-Seq is that sensitivity is proportional to cost, as sensitivity increases with the number of (expensive) parallel sequencing runs. To control for biases, ChIP-seq experiments often use the "input" as a control. This is DNA sequence resulting from the same pipeline as the ChIP-seq experiment, but omitting the immunoprecipitation step. It therefore should have the same accessibility and sequencing biases as the experiment data.
As with motif discovery, TF-binding sites search benefits from a comparative genomics approach. Searching a single genome for TFBS will yield very noisy results. If a number of related genomes are searched, then the search results can be compared and strengthened by requiring that a site be located, for instance, in the promoter region of the same gene for at least two or three species. As in the case of motif discovery, these methods are not often applied to verify experimental results, but can be used to guide experimental research. For instance, comparative genomics searches can be implemented to detect good candidate sites, which are then verified using an experimental technique.
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.
The calmodulin-dependent adenylate cyclase (Cya) domain of the cyclolysin toxin from Bordetella pertussis can be exploited as a reporter for translocation of effector proteins [PMID::10217833] [PMID::14702323]. Cya is not active in bacterial cytoplasm because bacteria do not possess calmodulin, and it is not naturally secreted or translocated by the type III secretion systems. However, when the N-terminal portion of an effector is fused to Cya, bacteria can deliver the resulting hybrid protein into the cytosol of host cells, where it can bind to calmodulin and produce cyclic AMP (cAMP) from ATP. Adenylate cyclase activity can also be assessed in vitro without translocation to a host.
An alternative method to ChIP-chip is DamID, which uses a methylase attached to the TF and a methyl-dependent PCR step to avoid the need for antibodies. The downside is that it only works in eukaryotes, because prokaryotes do methylate their adenosines (using, guess what, the Dam methylase).
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.
DNA-arrays (or DNA-chips or microarrays) are flat slabs of glass, silicon or plastic onto which thousands of multiple short single-stranded (ss) DNA sequences (corresponding to small regions of a genome) have been attached. After performing a mRNA extraction in induced and non-induced cells, the mRNA is again reverse transcribed, but here the reaction is tweaked, so that the emerging cDNA contains nucleotides marked with different fluorophores for controls and experiment. Targets will hybridize by base-pairing with those probes that resemble them the most. The array can then be stimulated by a laser and scanned for fluorescence at two different wavelengths (control and induced). The ratio or log-ratio between the two fluorescence intensities corresponds to the induction level.
The DNAse foot-printing method starts by focusing on a given region of interest (e.g. a promoter region) and amplifying it by PCR to obtain lots of sample. It then throws in the TF and then the DNAse. The mix is left to stir for a short time and then gel electrophoresis is run to compare the pattern of fragments in a control (no TF) and in the sample. If the TF has bound the sample, it will have protected a stretch of DNA (encompassing some fragments of the control) and thus those fragments will not appear in the sample gel. The fragments can then be cut-out from the gel, purified and sequenced to obtain the sequence of the protected region. This is often used to identify the binding motif of a TF for the first time. The foot-printing will typically resolve the protected region down to 50-100 bp, and the sequence can be then examined for possible TF-binding sites either by eye of using a computer search.
The Enzyme-linked immunosorbent assay (ELISA) is a test that uses antibodies and color change to identify a substance. In the context of transcriptional regulation, ELISA is sometimes used to identify a produced protein, especially if they are secreted proteins.
Electro-mobility shift-assays (or gel retardation assays) are a standard way of assessing TF-binding. A fragment of DNA of interest is amplified and labeled with a fluorophore. The fragment is left to incubate in a solution containing abundant TF and non-specific DNA (e.g. randomly cleaved DNA from salmon sperm, of all things) and then a gel is run with the incubated sample and a control (sample that has not been in contact with the TF). If the TF has bound the sample, the complex will migrate more slowly than unbound DNA through the gel, and this retarded band can be used as evidence of binding. The unspecific DNA ensures that the binding is specific to the fragment of interest and that any non-specific DNA-binding proteins left-over in the TF purification will bind there, instead of on the fragment of interest. EMSAs are typically carried out in a bunch of fragments, shown as multiple double (control+experiment) lanes in a wide picture. Certain additional controls are run in at least one of the fragments to ascertain specificity. In the most basic of these, specific competitor (the fragment of interest or a known positive control, unlabelled) is added to the reaction. This should sequester the TF and hence make the retardation band disappear, proving that the binding is indeed specific
Fluorescence anisotropy can be used to measure the binding constants and kinetics of reactions that cause a change in the rotational time of the molecules. If the fluorophore is bound to a small molecule, the rate at which it tumbles can decrease significantly when it is bound tightly to a large protein. If the fluorophore is attached to the larger protein in a binding pair, the difference in polarization between bound and unbound states will be smaller (because the unbound protein will already be fairly stable and tumble slowly to begin with) and the measurement will be less accurate. The degree of binding is calculated by using the difference in anisotropy of the partially bound, free and fully bound( large excess of protein) states measured by titrating the two binding partners.
A technique used to locate and isolate Fur sites via cloning. The genome of a target organism is digested with restriction enzymes, and ligated into high copy plasmids to create a plasmid library, which is then cloned into a cell line with a fur-repressed reporter. In cells where the introduced high copy plasmid contains a fur site, fur will be titrated away from it's binding site on the reporter (and throughout the genome), marking the colony. The cell can then be isolated, the plasmid extracted and sequenced for further study.
Genomic systematic evolution of ligands by exponential enrichment is a variation of SELEX that is restricted to actual genomic sequences (not randomly generated ones).
It should be always verified that the sequences reported are actually in the genome sequence.
In a GST pull-down assay, a gene of interest is fused with the GST gene, which codes for Glutathione S-transferase. Glutathione S-transferase has a high affinity for glutathione; consequently, the hybrid protein can be pulled down (purified) from cells using membranes coated with glutathione. If the gene of interest is bound to DNA, this DNA will be also pulled down and can be then analyzed by qPCR or sequencing.
This is a reporter assay technique, used typically to measure expression of a target gene. A promoter of interest is investigated by fusioning it with the reporter gene, which is then monitored. In GUS, the reporter is the β-glucuronidase enzyme from Escherichia coli, capable of transforming non-fluorescent substates into fluorescent, thereby allowing detection.
The HSQC experiment is a highly sensitive 2D-NMR experiment, where the transfer magnetization of a proton to a N or C isotope (the amide proton attached to a nitrogen in the peptide bond for proteins) is monitored by NMR, generating a specific peak in the spectrum. By analyzing the spectrum of a protein in the presence or absence of its cognate DNA binding site, chemical shifts in the spectrum can be detected that indicate binding.
Hydroxyl radical footprinting is almost identical to DNase footprinting, except that it uses hydroxyl radicals to digest non-protected DNA. While hydroxyl radicals yield much better resolution than DNases due to the considerable difference in their sizes, they are also harder and more time consuming to use, and thus this method is seen somewhat infrequently.
A specific antibody is used to isolate a protein from a mixture. Transcription factors isolated in this way can be incubated with a radiolabeled probe, and used to demonstrate binding.
Interferometric Reflectance Imaging Sensor (IRIS) is based on detecting changes in surface-reflected light due o the binding of ligands to biomolecules attached to the surface of a semi-transparent substrate. Differences in
the optical path lengths between a surface layer and a buried silica layers can be measured very precisely, which allows optical height information to be converted to accumulated mass (density) on the surface. For the purpose of TF-binding site identification, IRIS is coupled with DNA-array technology. DNA arrays with immobilized potential binding regions are exposed to the protein of interest, which is then eluted. Protein-bound fragments exhibit changes in reflectance that can be used to gauge binding quantitatively.
This technique is used to determine precise thermodynamic parameters such as the binding affinity of proteins and DNA. The temperature of two identical cells containing a known concentration of TF is monitored. The ligand (DNA) with a putative site or not is added to the cells in precisely measured aliquots. The difference in temperature observed after adding the ligand, because in one cell it binds to the target while in the other it does not, can be used to compute the energetics of the reaction and therefore the binding affinity of the TF for the DNA fragment.
Machine learning methods can be used to predict TF-binding sites, often using additional sequence-derived information (e.g. predicted DNA curvature), partitioning classical PSSMs into submotifs or computing correlations among site positions. These methods can yield improved predictions, but their efficiency must be properly assessed.
MALDI-TOF mass spectrometry is used to determine the mass of larger compounds such as proteins. It is a frequently used method of establishing protein identity when constructing a proteome.
Methidiumpropyl-EDTA (MPE) Fe(II) footprinting
This is a footprinting technique very similar to DNAse footprinting, which a synthetic molecule, methidium-propyl-EDTA (MPE), as the cleaving agent. MPE.Fe (II) footprinting appears more accurate than DNAse footprinting in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA.
In motif discovery, we are given a set of sequences that we suspect harbor binding sites for a given transcription factor. A typical scenario is data coming from expression experiments, in which we wish to analyze the promoter region of a bunch of genes that are up- or down-regulated under some condition. The goal of motif discovery is to detect the transcription factor binding motif (i.e. the sequence “pattern” bound by the TF), by assuming that it will be overrepresented in our sample of sequences. There are different strategies to accomplish this, but the standard approach uses expectation maximization (EM) and in particular Gibbs sampling or greedy search. Popular algorithms for motif discovery are MEME, Gibbs Motif Sampler or CONSENSUS. More recently, motif discovery algorithms that make use of phylogenetic foot-printing (the idea that TF-binding site will be conserved in the promoter sequences for the same gene in different species) have become available. These are not usually applied to complement experimental work, but can be used to provide a starting point for it. Popular algorithms include FootPrinter and PhyloGibbs.
The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mRNA) in a sample. Northern blotting involves the use of electrophoresis to separate RNA samples by size and detection with a hybridization probe complementary to part of or the entire target sequence.
A variation of DNAse footprinting in which certain residues are methylated to evidence their role in binding. Similar to site-directed mutagenesis in its ability to designate specific residues as being essential for binding.
Primer Extension Assay is a simple method of assessing expression levels. The total RNA produced by the culture is isolated. A short synthetic oligonucleotide primer (complementary to a short sequence on the target RNA) is radiolabelled (usually with 32P) and the primer anneals to the RNA. Reverse transcriptase then extends the RNA producing cDNA. The cDNA is analyzed on a polyacrylamide gel. The amount of cDNA in a band on the gel is proportional to the amount of initial RNA, thus providing expression levels of RNA from the culture.
Once the binding motif for a TF is known, this motif (which essentially defines a pattern) can be used to scan sequences in order to search for putative TF-binding site. This is useful, for instance, when trying to identify TF-binding site in ChIP-chip data. Searching for TF-binding site can be done in numerous ways. The most basic method is consensus search, in sequences are scored according to how many mismatches they have with the consensus sequence for the motif. A more elaborate way of searching involves using regular expressions, which allow to search for more loosely defined motifs [e.g. C(C/G)AT]. Common algorithms for this type of search include Pattern Locator and the DNA Pattern Find method of the SMS2 suite, but also some word processors. Finally, the mainstream way of conducting TF-binding site search is through the use of position-specific scoring matrices, which basically count the occurrences of each base at each position of the motif and use the inferred frequencies to score candidate sites. Algorithms in this last category include TFSEARCH, FITOM, CONSITE, TESS and MatInspector.
Quantitative PCR (or quantitative real time polymerase chain reaction) is a modification of the conventional PCR reaction used to amplify and simultaneously quantify a targeted DNA molecule. In its more standard incarnation, a inactive fluorescent dye is added to the PCR mix. This dye is activated upon binding double-stranded DNA; as the PCR iterates, there is more and more dsDNA and therefore more fluorescence intensity.
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.
Rapid Amplification of cDNA Ends is a technique to obtain the full length sequence of an RNA transcript. Most succinctly, the technique exploits the fact that PCR will stop at the ends of a mRNA. Basically, a primer mapping a "center" region of the mRNA is used to perform RT-PCR and terminal deoxynucleotidyl transferase (TdT) is used to add identical nucleotides to the 3' end of the cDNA. Standard PCR using another primer and the TdT-oligomer is performed in the other sense. The technique is frequently used to verify transcription start sites, which are relevant to the function (e.g. repression) of transcription factor binding sites.
Non-targeted mutagenesis. Typically accomplished in vivo by means of radiation or a DNA-damaging agent, or in vitro using degenerate PCR. Not frequently used in TF-binding site determination. It is sometimes used to investigate the effect of mutations in transcription factor binding domains, in conjunction with expression assays.
Similar to a PSSM search, a regular expression search looks for patterns in DNA, by making use of "degenerate" symbols and parenthetic constructs. For instance, [ATA (N:4–6) STGTC] indicates a pattern of ATA followed by 4 to 6 variable characters (N) and a suffix starting with a "strong" (S=G or C) base and the sequence TGTC.
A simplification of the Northern blot. The RNA is not first separated by electrophoresis. Instead, a mixture of RNA (e.g. a crude extract) is directly blotted onto a circular section of the matrix and hybridized with labeled DNA fragments.
In RNA-seq, RNA is extracted from the cell at a given time and reverse transcribed to obtain cDNA. This cDNA is then sequenced. This provides a snapshot of the "transcriptome" of an organism at a given time.
This is a technique used to detect typically mRNA with greater precision than Northern blot or RT-PCR. Therefore, it is commonly used to assess gene expression (specifically transcription). RNAse protection uses labelled RNA probes to bind desired targets. RNAses are then added to the mix and degrade all RNA that is not bound to probes. The remaining RNA is typically run on a gel to detect the size (and label) of the probe and determine which RNA it is.
This is a technique used to detect typically mRNA with greater precision than Northern blot or RT-PCR. Therefore, it is commonly used to assess gene expression (specifically transcription), even though it is a protection experiment. In S1 nuclease protection, extracted RNA is hybridized with complementary DNA probes and expose to S1 nuclease to degrade all RNA that is not bound to probes. The remaining (non-degraded) RNA is typically run on a gel to detect the size (and label) of the probe and determine which RNA it is.
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.
Surface plasmon resonance
Surface plasmon resonance (SPR) is a technique that exploits the generation of plasmons (waves) on the interface between a planar surface and vacuum/insulator. Plasmons are generated by an incident light beam. With proteins/DNA attached to the surface, ligands can be detected as changes in the SPR reflectivity, providing the means for accurate measurement of binding dynamics.
Survival in a particular environment/stress is used as a natural reporter, typically after knocking out an enzyme or regulator.
undecylprodigiosin (redD) reporter assay
A reporter assay in which a promoter of interest is cloned upstream of the Streptomyces redD gene, coding for the transcriptional activator of the biosynthetic pathway for undecylprodigiosin, a red-pigmented, mycelium-bound antibiotic made by Streptomyces coelicolor A3(2) and Streptomyces lividans. Promoter activity can be screened visually and spectrophotometrically for red pigmentation [PMID::11075931].
While DNase is by far the most commonly used tool for fragmenting DNA, it is not the only one. DNA footprinting can also be performed using other methods that cause non-specific breaks in the DNA, such as hydroxyl radicals, or UV radiation. There is little difference between these alternate methods of DNA cleavage and the more common DNase-mediated procedure aside from variant resolving power (hydroxyl radicals have a higher resolution) and small procedural changes. The data looks very similar, and is analyzed the same way.
Visual inspection of promoter region to identify putative binding sites based on previous knowledge
Western blot (quantitative) expression analysis
A Western blot (also frequently called immunoblot) is a gel-based technique used to determine the presence of specific proteins in a sample by means of separation (through gel electrophoresis) and detection (using a protein-specific antibody). Less frequent than the equivalent RNA Northern blot, it is typically used in TF-binding site analysis to validate differential regulation of target genes.
X-ray crystallography is a method to determine the molecular structure of a crystal that is typically used to probe at the tridimensional structure of proteins. In some cases, it is possible to co-crystallize the protein bound to its DNA binding site, providing detail on the particular arrangement of the two components. Doing so provides, obviously, unequivocal evidence of binding.
xylE reporter assay
A reporter assay based on the fusion of a promoter of interest with the xylE gene, which produces catechol dioxygenase, capable of converting the colorless substrate catechol to yellow hydroxymuconic semialdehyde.