Yeast Two-Hybrid System (and other cool stuff!)
Idea creds @Delphinidin.
The yeast two-hybrid system is a test used to determine if two proteins interact. The expression of a reporter gene indicates a positive result. Let's call the two proteins X and Y: protein X is conjugated to a DNA-binding domain (DBD) and protein Y is conjugated to an activator domain (AD). The DBD and AD, put together, function like a transcription factor (TF). The DBD does the "DNA-binding" part of a TF's function, and the AD does the "RNA polymerase-recruiting" part of a TF's function. If protein X interacts with protein Y, they will come in close proximity. Since each protein is conjugated to a component of a makeshift TF, if X and Y come together, so will the DBD and AD, forming a complete TF. If the proteins X and Y interact, and the "DBD+AD=functionally a TF" form, then transcription of the reporter gene occurs. The DBD binds to the promoter region, and the AD recruits RNA polymerase to transcribe the gene.
Another important note - the protein attached to the DBD (in this case, protein X), is known as the bait because it's conjugated to a stationary entity: the DBD just binds to the promoter and sticks there. On the other hand, the prey protein (protein Y), is bound to the mobile AD, which is attracted to the bait.
Note: does the "bait" and "prey" terminology sound familiar to you? Check this out (scroll to the bottom).
An example of a reporter gene is that for histidine synthesis. Before you continue reading, try to think about why this makes a good reporter gene, and come up with an experimental design using a histidine synthesis reporter.
Random image here so you don't see the answer immediately lol.
Did you think of auxotrophs? Auxotrophs, in this specific context, refer to mutant bacteria that need more than a minimal medium to survive; in other words, they need more than just sugars and basic stuff. For labs and USABO questions, it's important to know that auxotrophs need amino acid supplementation because they lack the equipment needed for biosynthetic pathways that create certain amino acids. In the case of using a histidine reporter gene, we need histidine auxotrophs: mutant bacteria that need histidine supplementation to survive because they can't make it themselves. This means that if the reporter gene for histidine synthesis is transcribed with the DBD+AD protein interaction (which would only happen if X and Y interact), then those bacteria would live. The rest would die. Thus, if bacteria survive, it means that histidine was synthesized, so X and Y must have interacted.
That's all I have for this week! Here's some more resources:
Good video explanation of yeast two-hybrid: https://youtu.be/w5Pvri4-cUA?si=MaMiilIIwnNj4rbu
CoIP - another method to determine protein interaction: https://www.sixfootscience.com/brain-snips/coip-how-to-find-if-two-proteins-are-acting-sus-with-each-other
FRET - another method to determine protein interaction: https://www.sixfootscience.com/brain-snips/dont-fret-fluorescence-resonance-energy-transfer-in-a-nutshell
