TEMA 2.2 Yeast Two-Hybrid System Overview

Podcast

Play an AI-generated podcast conversation about this lesson

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?

Detection of interactions in living cells (D) Signup and view all the answers

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

It recruits RNA polymerase II for transcription (B) Signup and view all the answers

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

They are critical for all cellular processes. (C) Signup and view all the answers

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

They can have separate DNA binding and activation domains. (C) Signup and view all the answers

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

It efficiently identifies in vivo protein interactions. (D) Signup and view all the answers

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

The bait and fish proteins interact successfully. (D) Signup and view all the answers

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

It identifies novel protein-protein interactions. (A) Signup and view all the answers

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

Fusion proteins may inhibit original interactions. (A) Signup and view all the answers

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

The bait and fish proteins are not interacting. (A) Signup and view all the answers

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

The test proteins are not localized to the nucleus. (B) Signup and view all the answers

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

Successfully measures enzymatic activity. (A) Signup and view all the answers

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

When interacting proteins successfully activate the reporter gene. (D) Signup and view all the answers

What role do chaperones play in protein interactions in yeast?

They facilitate the folding of proteins. (B) Signup and view all the answers

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

To study protein-protein interactions (C) Signup and view all the answers

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

1989 (A) Signup and view all the answers

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

Only the cDNA of the gene of interest (A) Signup and view all the answers

What are proteomics concerned with?

Evaluating the function and structure of proteins (D) Signup and view all the answers

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

It requires fewer resources for detection (D) Signup and view all the answers

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

Requires minimal quantities of proteins (A) Signup and view all the answers

Why is it important to study protein-protein interactions?

Understanding interactions helps elucidate protein function (C) Signup and view all the answers

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

Increased understanding of protein interaction networks (C) Signup and view all the answers

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

It interacts with a bait protein to trigger the activation domain. (D) Signup and view all the answers

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

It is used to determine if bait and fish proteins interact. (A) 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?

No interaction will be detected and the reporter gene will not be transcribed. (B) Signup and view all the answers

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

To assess the interaction with minimal nutrient interference. (D) Signup and view all the answers

What are the two domains of the Gal4 transcription factor?

Activating domain and binding domain. (C) Signup and view all the answers

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

To synthesize the RNA transcript from DNA. (D) Signup and view all the answers

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

The recombinant molecule lacks the necessary protein interactions. (C) Signup and view all the answers

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

There is successful interaction between bait and fish proteins. (A) 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.

Studying That Suits You

Use AI to generate personalized quizzes and flashcards to suit your learning preferences.

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.