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.

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.