Suppose you have a 1-kb segment of cloned DNA that is suspected to contain a eukaryotic promoter including a TATA box, a CAAT box, and an upstream GC-rich sequence. The clone also contains a gene whose transcript is readily detectable. Your laboratory supervisor asks you to outline an experiment that will (1) determine if eukaryotic transcription factors (TF) bind to the fragment and, if so, (2) identify where on the fragment the transcription factors bind. All necessary reagents, equipment, and experimental know-how are available in the laboratory. Your assignment is to propose techniques to be used to address the two items your supervisor has listed and to describe the kind of results that would indicate binding of TF to the DNA and the location of the binding. (Hint: The techniques and general results are discussed in this chapter.)

Key Concepts

Eukaryotic Promoters

Eukaryotic promoters are specific DNA sequences located upstream of a gene that facilitate the initiation of transcription. They typically contain essential elements such as the TATA box, which is crucial for the binding of transcription factors and RNA polymerase. Other elements, like the CAAT box and GC-rich sequences, enhance the efficiency of transcription. Understanding these components is vital for designing experiments to study transcription factor binding.

Transcription Factors (TFs)

Transcription factors are proteins that bind to specific DNA sequences, regulating the transcription of genes. They can act as activators or repressors, influencing the recruitment of RNA polymerase to the promoter region. Identifying the binding of TFs to DNA is essential for understanding gene expression regulation. Techniques such as electrophoretic mobility shift assays (EMSAs) or chromatin immunoprecipitation (ChIP) are commonly used to study TF binding.

Electrophoretic Mobility Shift Assay (EMSA)

EMSA is a technique used to study the binding of proteins, such as transcription factors, to specific DNA sequences. In this assay, a labeled DNA fragment is mixed with protein extracts, and the resulting complexes are separated by gel electrophoresis. The shift in mobility of the DNA-protein complex compared to free DNA indicates binding. This method allows researchers to determine if TFs bind to the promoter region and can be adapted to identify specific binding sites.