Yeast Two-Hybrid System - 2023 PDF

Dra. Macarena Rodríguez-Prados Valle 2023 Macarena Rodríguez-Prados Valle Methods and technics in biomedicine II Yeast transformation. Yeast two-hybrid Why yeast? Transformation conditions Plasmids lithium acetate technique Spheroplasts Biol...

Dra. Macarena Rodríguez-Prados Valle 2023 Macarena Rodríguez-Prados Valle Methods and technics in biomedicine II Yeast transformation. Yeast two-hybrid Why yeast? Transformation conditions Plasmids lithium acetate technique Spheroplasts Biolistic Glass bead methods. Yeast two-hybrid Principles Technique Advantages Limitations Macarena Rodríguez-Prados Valle  2. Yeast transformation. Yeast two-hybrid 1. MG3 (28/9/23) 2. MG4 (5/10/23) -Why yeast? Yeast two-hybrid Transformation conditions Principles Plasmids Technique Advantages lithium acetate technique Limitations Spheroplast 3. Flipped class-Group summary Biolistic (19/10/23) Group 5, 6 and 7. -Audiovisual resources (Jove video) 2. SM2 (5/10/22) -Questions Presentations/questions/videos Macarena Rodríguez-Prados Valle  The yeast two hybrid System - The technique was pioneered by Stanley Fields and OK-kyu Song in 1989. - It is an important technique used to study protein-protein interactions. - This procedure is based on the discovery that gene expression is S. cerevisiae depends on interactions between pairs of transcription factors of proteins. Macarena Rodríguez-Prados Valle Why do we need to study protein-protein interaction? Most proteins interact with other proteins for proper function, they must be studied in the context of their interacting partners to fully understand their function. Proteomics is the complete evaluation of the function and structure of proteins to understand an organism’s nature. As well as the study of the protein-protein interaction. Why yeast two Hybrid Method? Compared to classical biochemical approaches, which may require large amounts of purified proteins or good-quality antibodies, the two-hybrid system has minimal requirements to initiate detection, as only the cDNA of the gene of interest is needed. Macarena Rodríguez-Prados Valle - The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. - The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. - So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. Macarena Rodríguez-Prados Valle Screening Technologies for Protein-Protein Interactions - Protein-protein interactions are involved in all cellular processes. - Mapping of these interaction networks to elucidate the organization of the proteome into functional units is important for understanding the biological systems. - A large number of methods have been developed for screening protein interactions. The more classical biochemical approaches, such as copurification, affinity purification or coimmunoprecipitation of protein complexes require in vitro handling of protein extracts. Further limitations of these techniques include restricted sensitivity and bias towards high affinity interactions. - The Y2H technique allows detection of interacting proteins in living yeast cells. - Y2H can be easily automated for highthroughput studies of protein interactions on a genome-wide scale. Macarena Rodríguez-Prados Valle Based on transcription of DNA to RNA Macarena Rodríguez-Prados Valle Figure 1. The classical yeast two-hybrid system. (A) The protein of interest X, is fused to the DNA binding domain (DBD), a construct called bait. The potential interacting protein Y is fused to the activation domain (AD) and is called prey. (B) The bait, i.e. the DBD-X fusion protein, binds the upstream activator sequence (UAS) of the promoter. The interaction of bait with prey, i.e. the AD-Y fusion protein, recruits the AD and thus reconstitutes a functional transcription factor, leading to further recruitment of RNA polymerase II and subsequent transcription of a reporter gene. Macarena Rodríguez-Prados Valle  Yeast two-hybrid system - Yeast two-hybrid system is an ingenious way of detecting the protein-protein interaction in vitro. - Transcription factors have two domains, one for DNA binding and the other to allow binding to further proteins (activation domain). These occur as part of the same molecule in natural transcription factors; however, they may also be formed from two separate domains. - A recombinant molecule is formed encoding the protein under study as a fusion with the DNA-binding domain. It cannot however activate transcription. - Genes from a cDNA library are expressed as a fusion with the activator domain; this also cannot initiate transcription. Macarena Rodríguez-Prados Valle KEITH WILSON AND JOHN WALKER 2010 Yeast two-hybrid system Macarena Rodríguez-Prados Valle KEITH WILSON AND JOHN WALKER 2010  Yeast two-hybrid system: Protein-Protein interaction Transcription factors can bind to the promotor region of DNA and recruit the RNA polymerase and control and regulate the expression of each gene. Macarena Rodríguez-Prados Valle Macarena Rodríguez-Prados Valle 1- Yeast can metabolize galactose and they need enzymes for that like Gal gene and the transcription factor Gal4 - Gal4: Transcription factor with 2 domain - Gal gene Macarena Rodríguez-Prados Valle 2- Bait plasmid express a chimera of Gal4AD and bait protein and Fish plasmid express a chimera of Gal4 DBD and fish protein 3- The Gal4 activating domain and the DNA interaction domain will interact with each other only if bait and fish protein interact with each other. 4- The reporter gene will express only if bait and fish protein interact and allow to Gal4 activating and interaction domain Macarena Rodríguez-Pradosalso Valle interact. The reporter gene will be The reporter gene will not transcribed. be transcribed. Macarena Rodríguez-Prados Valle The reporter gene can be a metabolic gene like His reporter We can use a yeast strain unable to metabolize histidine and grow it in a histidine depleted medium. Then, the yeast colonies will only grow only if bait and fish proteins interact and the his reporter is produced. Macarena Rodríguez-Prados Valle Yeast co-transfected with Bait plasmid + plethora of fish plasmids How to read this data? If the colonies form, that means the histidine gene has been transcribed, that means the bait and the fish protein can interact with each other. Macarena Rodríguez-Prados Valle  Advantages of yeast two-hybrid system 1- Identifies novel protein-protein interactions. 2- It helps to identify protein cascades. 3- Identifies mutations that affect protein-protein interactions. 4- Determination of unknown protein function. Macarena Rodríguez-Prados Valle  Limitations of yeast two-hybrid system 1- A particular interaction may be absent in yeast (like if a bacterial protein is tested, the system may lack the chaperones important for folding or interaction). 2- If test protein are not localized to the nucleus, interacting proteins may show a negative result. 3- Fusion proteins may inhibit the original interaction. Macarena Rodríguez-Prados Valle Questions? Macarena Rodríguez-Prados Valle  References Principles and Techniques of Biochemistry and Molecular Biology Seventh edition EDITED BY KEITH WILSON AND JOHN WALKER, 2010. https://www.jove.com/es/v/3562/modified-yeast-two-hybrid-system-to-identify-proteins- interacting?list=h00KUa1V Genetic Transformation of Yeast Lin JS, Lai EM (2017) Protein-Protein Interactions: Yeast Two-Hybrid System.doi: 10.1007/978-1-4939-7033- 9_14 Macarena Rodríguez-Prados Valle Links of interest: Video 1: Yeast 2 hybrid system | Yeast two hybrid system for protein-protein interaction | Animated biology https://www.youtube.com/watch?v=Ypx42U5cmXE Video 2: Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin https://www.jove.com/es/v/3562/modified-yeast-two-hybrid-system-to-identify-proteins-interacting?list=h00KUa1V Video 3: 4.2: Yeast Two-Hybrid Assay to Determine Protein Self-Association in Yeast Cells Yeast Two-Hybrid Assay to Determine Protein Self-Association in Yeast Cells (jove.com) Video 4: 4.2: High-throughput Yeast Plasmid Overexpression Screen https://www.jove.com/v/2836/high-throughput-yeast-plasmid-overexpression-screen Macarena Rodríguez-Prados Valle