DNA footprint protection is a method that determines whether proteins bind to a specific sample of DNA and thus protect part of the DNA from random enzymatic cleavage by DNase I. A 400-bp segment of cloned DNA is thought to contain a promoter. The cloned DNA is analyzed by DNA footprinting to help determine if it has the capacity to act as a promoter sequence. The accompanying gel has two lanes, each containing the cloned 400-bp DNA fragment treated with DNase I to randomly cleave unprotected DNA. Lane 1 is cloned DNA that was mixed with RNA polymerase II and several TFII transcription factors before exposure to DNase I. Lane 2 contains cloned DNA that was exposed only to DNase I. RNA pol II and TFIIs were not mixed with that DNA before adding DNase I. Approximately what length is the DNA region protected by RNA pol II and TFIIs?

Verified step by step guidance

1. Understand the concept of DNA footprinting: DNA footprinting is a technique used to identify the specific region of DNA that is bound by a protein. When a protein binds to DNA, it protects that region from enzymatic cleavage by DNase I. By comparing the cleavage patterns of DNA with and without the protein, the protected region can be identified.
2. Examine the gel lanes: Lane 1 represents the DNA treated with RNA polymerase II and TFII transcription factors before DNase I cleavage. Lane 2 represents the DNA treated only with DNase I. The protected region will appear as a gap or absence of bands in Lane 1 compared to Lane 2, as the protein binding prevents DNase I from cleaving the DNA in that region.
3. Measure the protected region: To determine the length of the protected DNA region, compare the position of the gap in Lane 1 to the DNA ladder or size markers (if provided) on the gel. The size markers indicate the length of DNA fragments in base pairs (bp). The difference in fragment sizes between the start and end of the gap corresponds to the length of the protected region.
4. Account for the experimental setup: The DNA fragment used in this experiment is 400 bp long. The protected region will be a subset of this 400 bp fragment. Ensure that the gap in Lane 1 is consistent with the expected binding of RNA polymerase II and TFII transcription factors to the promoter region.
5. Estimate the length of the protected region: Based on the gel analysis, calculate the approximate size of the protected region by subtracting the size of the smaller fragment at the end of the gap from the size of the larger fragment at the start of the gap. This difference represents the length of the DNA region protected by RNA polymerase II and TFII transcription factors.

Key Concepts

DNA Footprinting

DNA footprinting is a technique used to identify the specific regions of DNA that are bound by proteins, such as transcription factors or RNA polymerase. When DNA is treated with DNase I, it cleaves unprotected regions, leaving a 'footprint' of protected areas where proteins are bound. This method allows researchers to visualize and analyze the binding sites of proteins on DNA, providing insights into gene regulation.

Transcription Factors and RNA Polymerase II

Transcription factors are proteins that bind to specific DNA sequences to regulate gene expression, often by recruiting RNA polymerase II, the enzyme responsible for synthesizing RNA from a DNA template. In the context of the experiment, RNA polymerase II and transcription factors (TFIIs) work together to initiate transcription at promoter regions, which are crucial for gene expression. Their binding can protect certain DNA regions from cleavage by DNase I.

Gel Electrophoresis

Gel electrophoresis is a laboratory technique used to separate DNA fragments based on their size. In the context of DNA footprinting, after DNase I treatment, the resulting DNA fragments are loaded onto a gel, and an electric current is applied. Smaller fragments move faster through the gel matrix, allowing researchers to visualize the protected regions as bands, which indicate where proteins have bound and prevented cleavage.