DNA footprint protection (described in Research Technique 8.1) 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?

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 enzymatic cleavage.

Promoter Regions

Promoter regions are specific sequences of DNA located upstream of a gene that signal the start of transcription. They contain binding sites for RNA polymerase and transcription factors, which are essential for the initiation of gene expression. In the experiment, the cloned 400-bp DNA segment is suspected to contain a promoter, and the length of DNA protected from DNase I cleavage indicates the extent of the promoter's interaction with RNA polymerase II and transcription factors.