A modification of the two-hybrid system, called the one-hybrid system, is used for identifying proteins that can bind specific DNA sequences. In this method, the DNA sequence to be tested, the bait, is fused to a TATA box to drive expression of a reporter gene. The reporter gene is often chosen to complement a mutant phenotype; for example, a HIS gene may be used in a his⁻ mutant yeast strain. A cDNA library is constructed with the cDNA sequences translationally fused to the GAL4 activation domain and transformed into this yeast strain. Diagram how trans-acting proteins that bind to cis-acting regulatory sequences can be identified using a one-hybrid screen.

Verified step by step guidance

1. Start by understanding the components of the one-hybrid system: the bait DNA sequence, the TATA box, the reporter gene, and the cDNA library fused to the GAL4 activation domain.
2. Diagram the setup: Place the bait DNA sequence upstream of the TATA box, which is linked to the reporter gene. This setup is integrated into the yeast genome.
3. Introduce the cDNA library: Each cDNA is fused to the GAL4 activation domain, creating a library of potential DNA-binding proteins.
4. Transform the yeast strain with the cDNA library: Each yeast cell receives a different cDNA-GAL4 fusion, allowing for the expression of various potential DNA-binding proteins.
5. Identify successful interactions: If a cDNA-encoded protein binds to the bait DNA sequence, the GAL4 activation domain will activate transcription of the reporter gene, allowing for selection based on the reporter gene's phenotype.

Key Concepts

One-Hybrid System

The one-hybrid system is a molecular biology technique used to identify proteins that bind to specific DNA sequences. It involves fusing a DNA sequence of interest (the bait) to a promoter, such as a TATA box, which drives the expression of a reporter gene. This system allows researchers to detect interactions between trans-acting proteins and cis-acting regulatory elements, facilitating the study of gene regulation.

Cis-acting and Trans-acting Elements

Cis-acting elements are DNA sequences located near a gene that regulate its expression, while trans-acting elements are proteins that bind to these sequences to influence gene activity. In the context of the one-hybrid system, the bait represents a cis-acting element, and the proteins from the cDNA library act as trans-acting factors that can bind to the bait, enabling the identification of specific protein-DNA interactions.

Reporter Gene

A reporter gene is a gene that encodes a protein easily measurable or observable, used to indicate whether a specific biological process has occurred. In the one-hybrid system, the reporter gene is linked to the bait DNA sequence, and its expression indicates successful binding of trans-acting proteins to the cis-acting elements. Common reporter genes include those encoding enzymes like HIS3, which can complement mutant phenotypes in yeast, allowing for straightforward detection of interactions.