Using Light to Control Gene Activity in Yeast

Light is an important part of our world. It affects how living things behave and grow. Different organisms use light in various ways to help them do things like grow, move, or even make food. A special group of proteins called photosensory proteins captures light. These proteins can change when they absorb light, leading to changes in how organisms react.

#What Are Photosensory Proteins?

Photosensory proteins are like little switches that turn on when they see light. They absorb light and go through a series of changes inside them. This change can trigger other actions within the organism. For example, a specific protein called EL222 can detect blue light. When it does, it changes shape and helps the organism turn on genes that might be needed in that moment.

#Structure of EL222

EL222 is made of two main parts. One part senses the light, and the other part does the work, like turning on a light switch in a room. The first part is called the sensory domain, which reacts to light, while the second part is the effector domain, which helps in binding to DNA, the instructions for building proteins.

In the dark, EL222 cannot bind to DNA because it has a shape that blocks it from doing so. When blue light shines on it, EL222 changes shape and can then bind to DNA, allowing it to activate specific genes. This process is reversible, which means it can turn on and off when light is available or not.

#How EL222 is Used in Science

Scientists have learned to use EL222 in labs to control genes in living cells. By shining a light on cells that have EL222, researchers can turn genes on or off. The light acts as a trigger, allowing them to study how genes work without changing anything else in the cell. This method is called optogenetics.

In some experiments, scientists modified EL222 by adding new parts to it. They created a version called VP-EL222, which can move into the nucleus of cells, where DNA is located, and activate genes when light is present. This modified version has been widely used in research.

#Working with Yeast

One popular organism for these experiments is yeast. Yeast is easy to grow in a lab and can be used to understand many basic biological processes. Scientists have used VP-EL222 in yeast to study how certain genes are controlled under different conditions, like the presence of nutrients.

For example, when yeast has enough phosphate, a nutrient, certain genes are turned off. But when phosphate is low, those genes are activated. By using light and the VP-EL222, researchers can turn on these genes to study how yeast responds to different phosphate levels.

#Phosphate and Gene Control

The PHO5 gene in yeast is responsible for breaking down phosphate. When phosphate is plenty, the gene is off because the yeast doesn't need to produce more of the enzyme that breaks it down. However, when phosphate levels drop, the yeast must turn on the PHO5 gene to absorb what it needs.

The way yeast turns on this gene involves several steps. First, a protein called Pho4 must enter the nucleus and bind to specific sites on the PHO5 gene. When phosphate is low, Pho4 gets activated and brings along helpers to turn the gene back on. This process is complex and involves changes in the structure of DNA and proteins within the cell.

#What Scientists Did

Researchers took the VP-EL222 system and added it to the yeast. By shining blue light on the yeast, they could turn on the PHO5 gene even when phosphate was available. They also looked at another gene, PHO84, which also responds to phosphate. Understanding these processes helps scientists learn how cells react to their environment.

In their experiments, the scientists combined VP-EL222 with different proteins to see if they could improve its ability to control these genes. They created different versions of EL222, some of which included larger proteins. This would help determine how much additional cargo EL222 could carry without losing its ability to work effectively.

#Using CRISPR for Gene Editing

To make their experiments more effective, scientists used a tool called CRISPR. This technology allows them to edit genes in the yeast’s DNA directly. They inserted the binding site for VP-EL222 close to the PHO5 and PHO84 genes so that when light was applied, they could easily turn on these genes.

Through the process, they discovered that adding VP-EL222 did not change how the PHO5 and PHO84 genes normally behaved. They continued to express the genes as needed, depending on the phosphate levels present. This means that using EL222 didn’t interfere with how yeast naturally responds to phosphate.

#Measuring Gene Activity

To see how well the genes were being turned on, the researchers measured the levels of RNA produced by these genes. RNA is the molecule that tells the cell how to make proteins. By quantifying the RNA, they could determine if the genes were being expressed properly in response to light.

The scientists found that when yeast was illuminated with blue light, the PHO5 gene produced a significant amount of RNA compared to when it was in the dark. The level varied based on the phosphate availability. In high phosphate conditions, they observed less RNA production overall, while low phosphate conditions allowed for much more gene activity.

#The Role of Additional Proteins

In addition to VP-EL222, the researchers also tested other proteins that could link up with EL222 to further manipulate gene expression. They created fusions of EL222 with different proteins, including transcriptional activators and repressors. Activators help turn on genes, and repressors help turn them off.

Some of their experiments involved proteins that are already part of the yeast system. By attaching them to EL222, the scientists aimed to create a system that was more efficient and reflective of the natural biology of yeast. They found that while some combinations worked well, others did not function as desired, highlighting the complexity of working with these systems.

#Results from Experiments

After numerous tests, the researchers concluded that different combinations of EL222 and its partners showed varying effectiveness in controlling gene expression. VP-EL222 alone could effectively activate the PHO5 gene in low phosphate conditions but struggled somewhat in high phosphate scenarios.

In contrast, attaching a native yeast protein like Pho4 to EL222 allowed for much clearer control over the PHO5 gene. This means that using native proteins can sometimes yield better results than using synthetic ones.

The results also showed that the positioning of the binding sites relative to the gene itself plays a critical part in the system’s efficiency. The closer the binding site to the gene, the more effective the light-induced activation would be.

#Observations on Gene Repression

In addition to activating genes, researchers tested whether they could successfully repress gene activity using EL222. They created a fusion of EL222 with a corepressor protein, Ume6, which is typically involved in turning off genes during specific conditions.

Using this new construct, they found that they could lower the expression of the PHO5 gene even under low phosphate conditions. This ability to repress gene expression adds another layer to how researchers envision controlling gene activity in yeast and potentially other organisms.

#Conclusion

The work done with EL222 and light manipulation provides valuable insight into how gene expression can be controlled in living organisms. By using light as a trigger, scientists can study complex biological processes in a controlled manner.

The ongoing refinement of these techniques aids researchers in uncovering fundamental biological principles that govern how cells respond to their environment. This understanding has the potential for wider applications in biotechnology, synthetic biology, and medicine, as targeted gene control can lead to innovations in how we design organisms for specific tasks.

Studies like these showcase the intertwined relationship of proteins, DNA, light, and the environment, and how together they form the basis of life’s intricate dance.