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Before tackling the topic of DNA footprinting, we need to look at some essential aspects of RNA synthesis i.e. transcription.
When DNA is converted into RNA, the process is called DNA transcription. Initiation of RNA synthesis does not take place at random points in a DNA molecule. Instead, the RNA polymerase enzyme binds to specific DNA sequences, called promoters, which direct the transcription of adjacent segments of DNA. The DNA sequences where RNA polymerases bind can be quite variable.
A process that provides information about the interface between RNA polymerase and promoters is called DNA footprinting. It is a technique that identifies the DNA sequences bound by particular proteins.
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DNA Footprinting Procedure
- A DNA fragment thought to contain sequences recognized by a DNA-binding protein is isolated and radio labeled at one end of one strand.
- Once the DNA fragments are labeled, they are mixed in a test tube with a DNA-binding protein and DNAse.
- In a separate test tube, same labeled DNA with DNAse is mixed without the DNA-binding protein. This is for comparison.
- DNAse is a cleavage agent which cuts the DNA in both samples. No cuts are made in the area where RNA polymerase has bound. It has protected the DNA.
- Only labeled strands are detected in next step.
- Separation of fragments is done by polyacrylamide gel electrophoresis, and visualized by autoradiogram.
- Missing bands (hole or footprint) indicate where RNA polymerase was bound to DNA.
Binding of proteins to DNA sequences both near to and distant from the promoter can also affect levels of gene expression. Protein binding activates transcription by facilitating RNA polymerase binding or facilitating steps further along in the initiation process or it can repress transcription by blocking the activity of the polymerase.
DNA footprinting helps identify the promoter region and thereby the gene expression level.
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DNA Footprinting Applications in Research
DNA footprinting is often used to identify the binding sites of proteins in a DNA molecule. Researchers often use this technique to identify whether a particular protein can activate or inhibit transcription. In addition, scientists also use this method to detect where proteins bind to DNA in a living cell. In this kind of experiment, scientists often grow cells under artificial conditions where the protein of interest would be more likely to bind to DNA. The resulting DNA-protein complexes are then purified from the cell, and the DNA sequences are identified.
Since DNA footprinting is being used to identify DNA sequences where proteins bind to DNA, it is of great importance in genetic research, especially in work coming from the Human Genome Project. It enables researchers to identify many functional genes present in large human DNA sequences.