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. Understand the key components of the one-hybrid system: the bait DNA sequence (a cis-acting regulatory element) fused upstream of a minimal promoter such as a TATA box, which controls the expression of a reporter gene (e.g., HIS gene).
2. Recognize that the reporter gene is integrated into a yeast strain with a mutant phenotype (e.g., his⁻), so expression of the reporter gene can restore the wild-type phenotype, allowing growth on selective media.
3. Construct a cDNA library where each cDNA is fused to a known transcriptional activation domain (such as the GAL4 activation domain), creating fusion proteins that can potentially bind to the bait DNA sequence if they recognize it.
4. Transform the yeast strain containing the bait-reporter construct with the cDNA library plasmids, so that each yeast cell expresses a different fusion protein from the library.
5. Select for yeast cells that grow on selective media lacking histidine; these cells express the reporter gene because the fusion protein binds the bait DNA sequence, activating transcription. These positive clones identify trans-acting proteins that specifically bind the cis-acting bait sequence.

Key Concepts

One-Hybrid System

The one-hybrid system is a molecular biology technique used to identify proteins that bind specific DNA sequences. It involves fusing a DNA sequence of interest (bait) upstream of a reporter gene, which is activated only when a protein binds the bait. This method helps detect DNA-protein interactions by linking binding events to reporter gene expression.

Cis-acting and Trans-acting Elements

Cis-acting elements are DNA sequences, like promoters or enhancers, that regulate gene expression on the same DNA molecule. Trans-acting factors are proteins, such as transcription factors, that bind these cis-elements to modulate transcription. Identifying trans-acting proteins that bind specific cis-elements is key to understanding gene regulation.

Reporter Gene and Selection in Yeast

Reporter genes produce easily measurable products, allowing detection of protein-DNA interactions. In yeast one-hybrid assays, reporter genes like HIS complement mutant strains lacking histidine synthesis, enabling growth only if the reporter is activated. This selection links protein binding to survival, facilitating identification of DNA-binding proteins.