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. What additional genetic experiments would you suggest to verify that this region of cloned DNA contains a functional promoter?

Verified step by step guidance

1. Step 1: Perform a reporter gene assay. Clone the 400-bp DNA segment upstream of a reporter gene (e.g., luciferase or GFP) in a plasmid vector. Introduce this plasmid into a suitable host cell and measure the expression of the reporter gene. If the DNA segment contains a functional promoter, it will drive the expression of the reporter gene, resulting in detectable activity (e.g., fluorescence or luminescence).
2. Step 2: Conduct a mutational analysis. Introduce specific mutations or deletions into the 400-bp DNA segment to identify critical regions required for promoter activity. Test the mutated sequences using the reporter gene assay to determine how the mutations affect gene expression.
3. Step 3: Perform an electrophoretic mobility shift assay (EMSA). Use labeled DNA fragments from the 400-bp region and mix them with nuclear extracts or purified transcription factors. If the DNA contains a functional promoter, specific protein-DNA complexes will form, causing a shift in the mobility of the DNA during gel electrophoresis.
4. Step 4: Test for transcription initiation in vitro. Use an in vitro transcription assay with the 400-bp DNA segment as a template. Add RNA polymerase II and necessary transcription factors to the reaction and analyze the RNA products to confirm whether transcription is initiated from this DNA region.
5. Step 5: Analyze chromatin accessibility using DNase I hypersensitivity or ATAC-seq. Functional promoters are often located in regions of open chromatin. Test whether the 400-bp DNA segment is in an accessible chromatin state, which would support its role as a promoter.

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. By treating DNA with DNase I, which cleaves unprotected DNA, researchers can visualize the protected regions on a gel. This method helps determine the binding sites of proteins and can indicate the presence of functional elements like promoters.

Promoter Functionality

A promoter is a DNA sequence that initiates transcription of a gene by providing a binding site for RNA polymerase and transcription factors. To verify a region's functionality as a promoter, experiments can assess its ability to drive gene expression in a cellular context, often using reporter assays that measure the activity of a reporter gene linked to the promoter of interest.

Transcription Factors and RNA Polymerase II

Transcription factors are proteins that bind to specific DNA sequences to regulate gene expression, often working in conjunction with RNA polymerase II, which synthesizes mRNA from DNA. The presence of these factors is crucial for the activation of promoters. Experiments that manipulate the levels or activity of these factors can help confirm whether a DNA region functions as a promoter by observing changes in transcription levels.