TEMA 2.2 Yeast Two-Hybrid System Overview
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TEMA 2.2 Yeast Two-Hybrid System Overview

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Questions and Answers

What is a primary limitation of classical biochemical approaches for protein interaction screening?

  • Automatic high-throughput screening capabilities
  • Identification of protein interactions in vivo
  • High sensitivity in detecting low affinity interactions
  • In vitro handling of protein extracts (correct)

    • Which feature of the yeast two-hybrid system enhances its capability for high-throughput studies?

  • Limited sensitivity to high affinity interactions
  • Automation for genome-wide scale interactions (correct)
  • Requirement for in vitro protein handling
  • Use of mass spectrometry for analysis

    • In the yeast two-hybrid system, what does the bait refer to?

  • The DNA binding domain fused protein (correct)
  • The protein that does not interact with the target
  • The activation domain fused protein
  • The RNA polymerase II enzyme

    • What is a significant advantage of the yeast two-hybrid (Y2H) screening method?

    <p>Detection of interactions in living cells</p> Signup and view all the answers

    What role does the activation domain (AD) play in the yeast two-hybrid system?

    <p>It recruits RNA polymerase II for transcription</p> Signup and view all the answers

    Which of the following statements about protein-protein interactions is true?

    <p>They are critical for all cellular processes.</p> Signup and view all the answers

    What is an essential characteristic of transcription factors mentioned in relation to protein interactions?

    <p>They can have separate DNA binding and activation domains.</p> Signup and view all the answers

    Why has the yeast two-hybrid method become dominant in published interaction studies?

    <p>It efficiently identifies in vivo protein interactions.</p> Signup and view all the answers

    What does the successful growth of yeast colonies indicate in this system?

    <p>The bait and fish proteins interact successfully.</p> Signup and view all the answers

    Which advantage is associated with the yeast two-hybrid system?

    <p>It identifies novel protein-protein interactions.</p> Signup and view all the answers

    Which of the following is a limitation of the yeast two-hybrid system?

    <p>Fusion proteins may inhibit original interactions.</p> Signup and view all the answers

    What is suggested if the histidine gene is not transcribed in the experiment?

    <p>The bait and fish proteins are not interacting.</p> Signup and view all the answers

    What is one possible reason for a negative result when testing a protein interaction in yeast?

    <p>The test proteins are not localized to the nucleus.</p> Signup and view all the answers

    Which of the following does NOT represent an advantage of the yeast two-hybrid system?

    <p>Successfully measures enzymatic activity.</p> Signup and view all the answers

    How do yeast colonies grow in the two-hybrid system?

    <p>When interacting proteins successfully activate the reporter gene.</p> Signup and view all the answers

    What role do chaperones play in protein interactions in yeast?

    <p>They facilitate the folding of proteins.</p> Signup and view all the answers

    What is the primary purpose of the yeast two-hybrid system?

    <p>To study protein-protein interactions</p> Signup and view all the answers

    Which year was the yeast two-hybrid technique first introduced?

    <p>1989</p> Signup and view all the answers

    What is needed to initiate detection in the two-hybrid system?

    <p>Only the cDNA of the gene of interest</p> Signup and view all the answers

    What are proteomics concerned with?

    <p>Evaluating the function and structure of proteins</p> Signup and view all the answers

    What is a significant advantage of using the yeast two-hybrid method over classical biochemical techniques?

    <p>It requires fewer resources for detection</p> Signup and view all the answers

    Which of the following is NOT a limitation of the yeast two-hybrid system?

    <p>Requires minimal quantities of proteins</p> Signup and view all the answers

    Why is it important to study protein-protein interactions?

    <p>Understanding interactions helps elucidate protein function</p> Signup and view all the answers

    What will the advancement of high-throughput methods for interactomics research lead to?

    <p>Increased understanding of protein interaction networks</p> Signup and view all the answers

    What role does the Gal4 transcription factor play in the yeast two-hybrid system?

    <p>It interacts with a bait protein to trigger the activation domain.</p> Signup and view all the answers

    Which statement accurately describes the role of the reporter gene in the yeast two-hybrid system?

    <p>It is used to determine if bait and fish proteins interact.</p> Signup and view all the answers

    In the context of the yeast two-hybrid system, what happens if the bait and fish proteins do not interact?

    <p>No interaction will be detected and the reporter gene will not be transcribed.</p> Signup and view all the answers

    What is the purpose of using a yeast strain unable to metabolize histidine in this system?

    <p>To assess the interaction with minimal nutrient interference.</p> Signup and view all the answers

    What are the two domains of the Gal4 transcription factor?

    <p>Activating domain and binding domain.</p> Signup and view all the answers

    What is the function of the RNA polymerase in gene expression?

    <p>To synthesize the RNA transcript from DNA.</p> Signup and view all the answers

    Why is transcription initiation not achieved solely by forming a recombinant molecule?

    <p>The recombinant molecule lacks the necessary protein interactions.</p> Signup and view all the answers

    What is implied when the expression of the reporter gene occurs?

    <p>There is successful interaction between bait and fish proteins.</p> Signup and view all the answers

    Study Notes

    Yeast Two-Hybrid System

    • A technique used to study protein-protein interactions
    • The yeast two-hybrid system uses reporter genes, a bait plasmid and a fish plasmid
    • The GAL4 gene is used for expression of the reporter gene.
    • The bait plasmid is comprised of a chimeric protein: the GAL4AD domain and a bait protein.
    • The fish plasmid is comprised of a chimeric protein: the GAL4DBD domain and a fish protein.
    • The interaction between the bait and fish proteins will result in the GAL4AD domain and the GAL4DBD domain interacting.
    • If the GAL4AD domain and the GAL4DBD domain interact, the reporter gene will be transcribed.
    • Using a reporter gene to track protein interactions is a common technique, like using a his reporter gene.
    • The his reporter gene is used in a yeast strain lacking the ability to metabolize histidine.
    • This yeast strain can be grown in a histidine depleted medium.
    • The reporter gene is expressed if the bait and fish proteins interact and allow the GAL4AD and GAL4DBD domains to interact.

    Advantages of the Yeast Two-Hybrid System

    • Identifies novel protein-protein interactions
    • Identifies protein cascades
    • Identifies mutations that affect protein-protein interactions
    • Determination of unknown protein function

    Limitations of the Yeast Two-Hybrid System

    • A particular interaction may be absent in yeast
    • If test proteins are not localized to the nucleus, interacting proteins may show a negative result
    • Fusion proteins may inhibit the original interaction

    Why Study Protein-Protein Interaction?

    • Proteins often need to interact with other proteins to function.
    • Studying proteins in the context of their interacting partners helps understand their function.
    • Studying protein-protein interactions is a key aspect of proteomics.

    Why the Yeast Two-Hybrid Method?

    • Requires less preparation compared to other methods of studying protein interactions.
    • Only needs the cDNA of the gene of interest.
    • Can be automated for high-throughput studies.
    • Can be used to screen protein interactions on a genome-wide scale.

    Yeast Two-Hybrid System and Transcription

    • The yeast two-hybrid system relies on the transcription of DNA to RNA.
    • GAL4 is a transcription factor that binds the upstream activator sequence (UAS) of a promoter.
    • The bait protein, fused to the GAL4DBD domain, binds the UAS.
    • The fish protein, fused to the GAL4AD domain, binds to the bait protein.
    • This interaction reconstitutes a functional transcription factor.
    • The functional transcription factor recruits RNA polymerase II to the UAS, leading to transcription.

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    Yeast Two-Hybrid System - 2023 PDF

    Description

    This quiz covers the yeast two-hybrid system, a crucial technique for studying protein-protein interactions. It examines the roles of bait and fish plasmids, as well as the GAL4 gene in driving reporter gene expression. Dive into the mechanism of how these proteins interact and what it means for understanding biological processes.

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