Locating Transcription Factors

Overview 
This post is going to cover a bit about locating the transcription factors, purifying the protein, and then running some tests in vitro and in vivo to prove that yes, this protein really does aid in initiating transcription.

Particular sequences on DNA, known as transcriptional control elements, enhance transcription. They are called enhancers. Regulatory proteins called transcription factors bind to the enhancers, helping to initiate transcription of a gene. However, for this post we are not too concerned about the process of initiating transcription. Our goal is to identify and isolate these proteins (transcription factors) in the cell. How do we do this? There are three different methods that I know of (probably more exist) which shall be discussed. These techniques can be used together to purify the transcription factors desired.

1) Chromatography. 
The basic idea behind the chromatography technique is that you pass a sample solvent of various proteins through a column of solid chromatography matrix and collect the protein in a test tube below after a certain time has elapsed. Different proteins will travel through the solid matrix at different rates depending on their properties. Here are three major types of chromatography used.

a) Ion-Exchange Chromatography
The solid matrix in this case is made up of positively charged beads. As the solvent passes through the matrix, the negatively-charged proteins will bind to the positively charged beads, and hence, will pass through the column more slowly. The negatively charged proteins will emerge first. To remove the positively charged proteins from the matrix, a salt is applied. The negative salt ions will bind to the positively charged matrix, displacing the negatively charged proteins.
* separation by charge

b) Gel-Filtration Chromatography
This time the solid matrix consists of porous beads which trap the smaller proteins, retarding them through the column. The large proteins pass through unhindered. The smaller proteins will eventually come out at the other end, a longer time will have just elapsed.
* separation by size

Step a) and b) can be done in interchangeable order, but step c) is always the last step.

c) Affinity Chromatography
This type of chromatography is the most specific type of chromatography. The beads are marked with a covalently attached sequence (or substrate in the case of an enzyme) specific to the protein which will bind and trap the desired protein molecule as it passes through the matrix. The rest of the proteins will pass through. To extract the desired protein, the nuclear extract is incubated with the beads, washed, and then eluted with increasing salt concentration. This method gives the highest purity of the protein.

2) DNase I Footprinting Experiments
These are useful as they reveal specific binding sites for DNA binding proteins and can be used as an assay in transcription factor purification. The DNA is end-labeled (as a point of reference) and then is partially digested with a nuclease like a restriction enzyme that cuts the DNA randomly. We take a strand of DNA without the DNA-binding protein and, the restriction enzyme will cut wherever it pleases creating numerous fragments. However, when we let the DNA-binding protein bind to its binding site on the DNA, then the restriction enzyme will cut everywhere except where the protein is bound. This allows you to know the protein's specific sequence. Now, if we pass this sample through an electrophoresis gel, the place where the protein bound will have no lines, because there will be no fragments at those base pairs.

3) EMSA (gel-shift, or band-shift assay) 
The EMSA technique is better than footprinting for quantitative analysis of DNA-binding proteins, as to whether they bind or not to the DNA. However, it does not provide the specific DNA-binding sequence. When the DNA is passed through the gel, it is radiolabelled and contains a known regulatory element. The control is called a probe, and it will have a bound protein to its DNA fragment. The bound protein will cause the probe to migrate more slowly through the gel than DNA alone. To find whether the protein has bound to other strands of DNA, a band will show on the gel, whereas unbound DNA will pass through the gel quickly to the other side. This test is done with different DNA sequences from the same chromosome and the same proteins to find the binding sequence, although the specific base pairing cannot be isolated from this technique.

A Note about Transcription Factors 
Transcription factors are proteins that stimulate or repress transcription by binding to promoter-proximal elements (a type of enhancer close to the promoter binding site, the TATA box), enhancers, and repressors in eukaryotic DNA. The modular protein structure of a transcription factor has a single DNA binding domain and one or more activation domains or repression domains, depending on whether the transcription factor is an activator or a repressor respectively.

Now that we have purified the protein, we need to prove that it does aid in initiating transcription indeed.

1) In Vitro Assay for Transcription Activity 

In an example using SP1 as the transcription factor and Adenovirus DNA and SV40 DNA, Adenovirus is used as the control, as it does not require SP1 to initiate transcription. With or without SP1, transcription will occur. SV40, however, will not have any transcription if SP1 is not present. If SP1 is provided, transcription happens. Transcription is shown on these tests as a black band.

2) In Vivo Assay for Transcription Activity 

This test is more powerful than the above in vitro assay because if something happens in a test tube doesn't mean it necessarily happens in the cell. Inserted into the cell, is a plasmid 1 containing the sequence required for the cell to transcribe the protein transcription factor being tested. Also, a plasmid 2 is inserted containing the binding sequence for the transcription factor along with reporter gene, which is just a gene which has an obvious effect upon the cell when transcribed. This is a co-transfection assay. If the transcription factor is truly a transcription factor, the reporter gene will be expressed in the cell. The assay can identify repressors as well as activators, and is a necessary method in cells that lack or do not express the gene encoding the protein to be tested.

A Note about Transcriptional Activators

Activators are modular proteins that have distinct functional domains including a DNA binding domain and an activation domain, which interacts with other proteins to stimulate transcription.

Activators were demonstrated in an experiment where two plasmids were again inserted into a cell. A variety of lengths of sequences for the gene that contained information to encode the transcription factor GAL4 in plasmid 1 were taken. Then it was measured whether or not the LacZ reporter gene was transcribed from the plasmid 2.

This example is a little bit trippy, because we are measuring the effects of transcription by how the cell transcribed the inserted plasmid 1 sequence of the transcription factor by (wait for it), measuring the transcription factor's initiation of transcription of the reporter gene in plasmid 2. Phewf!

The results examined were the binding to UASgal enhancer site on plasmid 2, and the B-galactosidase activity in the cell. When neither was shown in the deletion of base pair for the sequence of GAL4, the conclusion was that that was a DNA binding domain. No DNA binding domain present meant no binding to the enhancer site was possible, hence no transcription. However, with subsequent deletion of bases in plasmid 1, binding to UASgal was found, but little to no B-galactosidase activity, demonstrating the activation domains. This was significant because it revealed the activation domains to be in separate locations on the coding sequence than the DNA binding domain.

Activation domains can be in front of or behind the DNA binding domain. There can be multiple activation domains, but there is always only one DNA binding domain. If the domains are swapped around or fused together, a functional protein still results. They don't seem to be too fussy.

A Note About Transcriptional Repressors

As you might guess, they are the functional converse of activators. Most are modular proteins with a DNA-binding domain and a repression domain, which function by interacting with other proteins.