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. Explain why this gel provides evidence that the cloned DNA may act as a promoter sequence.

Verified step by step guidance

1. Understand the concept of DNA footprinting: DNA footprinting is a technique used to identify the specific regions of DNA that are bound by proteins. When a protein binds to a DNA sequence, it protects that region from enzymatic cleavage by DNase I, leaving a 'footprint' on the gel where no cleavage occurs.
2. Analyze the experimental setup: Lane 1 contains DNA mixed with RNA polymerase II and transcription factors (TFIIs) before DNase I treatment, while Lane 2 contains DNA treated only with DNase I, without any proteins added. The purpose of this comparison is to determine if the proteins bind to the DNA and protect specific regions.
3. Interpret the gel results: In Lane 1, if a region of the DNA is protected from DNase I cleavage, it will appear as a gap or absence of bands on the gel. This indicates that RNA polymerase II and TFIIs are binding to that specific region of the DNA. In Lane 2, the DNA is fully exposed to DNase I, so cleavage occurs randomly, resulting in a continuous ladder of bands.
4. Relate the results to promoter activity: Promoters are specific DNA sequences where RNA polymerase and transcription factors bind to initiate transcription. If a protected region is observed in Lane 1 but not in Lane 2, it suggests that RNA polymerase II and TFIIs are binding to that region, indicating that the DNA segment may contain a promoter sequence.
5. Conclude the evidence: The presence of a protected region in Lane 1 provides strong evidence that the cloned DNA contains a sequence capable of binding RNA polymerase II and transcription factors, which is a key characteristic of a promoter. This supports the hypothesis that the cloned DNA may act as a promoter sequence.

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 behind a 'footprint' of protected areas where proteins are bound. This method allows researchers to visualize the binding sites and infer the functional roles of these regions in gene regulation.

Promoter Sequences

Promoter sequences are specific regions of DNA that initiate transcription of a gene. They are typically located upstream of the coding region and are recognized by RNA polymerase and transcription factors. The presence of a promoter is indicated by the binding of these proteins, which protects the DNA from cleavage, suggesting that the region is functionally important for gene expression.

Transcription Factors and RNA Polymerase II

Transcription factors are proteins that bind to specific DNA sequences to regulate the transcription of genes. RNA polymerase II is the enzyme responsible for synthesizing mRNA from DNA during transcription. The interaction between RNA polymerase II, transcription factors, and the promoter region is crucial for the initiation of transcription, and their presence in the experimental setup indicates that the cloned DNA may function as a promoter.