WCRISPR may be the most remarkable genetic technology to come out of microbes. Many of the tools researchers use to control genes and their products are borrowed from bacterial systems. One of them is rack The operon regulates the expression of genes involved in lactose metabolism. Escherichia coli. Applications from the bench to the industrial vat use it to selectively turn on gene expression.
However, in most cases, researchers borrow only part of it. rack operon system. The researchers used the repressor protein LacI, which physically binds to DNA upstream of gene promoters and blocks RNA polymerase, and added an inducible molecule that removes LacI when researchers want to turn on gene expression. I will. This engineered system regulates gene expression, which requires: high concentration Control LacI to consistently suppress them.1 By comparison, the complete form of the operon that occurs in the following cases is Escherichia coli It contains an additional binding site for LacI that forms a DNA loop and represses LacI transcription more efficiently. rack The use of repressor proteins in operon genes is reduced.
One team is interested in exploiting this capability to develop new approaches to gene regulation. In a study published in Nucleic acid researchMayo Clinic researchers have created a novel protein that mimics the double-binding ability of LacI, providing a new approach to future gene expression control.2
LacI is a tetrameric protein that uses two dimers to bind to two specific identical DNA sequences. This specificity limits the possibilities for using the LacI system when researchers are unable to insert this sequence and has prompted the search for more flexible repressor proteins that can recognize more regions of DNA.
“It was probably around 2015 that we started working on ideas like designer gene silencing loops.” nicole beckera molecular biologist at the Mayo Clinic and co-author of the study.
TALE proteinTranscription activator-like effector (TALE) is a class of proteins derived from plant pathogens. Xanthomonas Genus.3 TALE recognizes DNA using repeating 34-amino acid regions that include 12-amino acid regions.th and 13th The amino acids determine the nucleic acid that each segment binds to. Pathogens use these proteins to activate plant gene expression and promote bacterial survival, but scientists cracked this DNA binding code Create a custom TALE that can recognize any sequence you want to target.4,5
In this study, the team copied the two-headed nature of LacI by linking two different TALE dimers called A and O.2recognizes different DNA sequences. They inserted these two sequences upstream of a reporter gene and used a colorimetric assay to determine whether the protein repressed expression of the reporter based on the lack of color. Their goal was to determine the parameters required to achieve gene silencing comparable to LacI.
First, the research team evaluated the effect of the order of the two TALE dimers within the protein on repression efficiency. Previous research has shown that placing more powerful repressors further away Because the promoter improved repression, we tested the degree of repression of A and O.2.6 Next, we inserted the stronger TALE A sequence into the distal region of the promoter and the weaker TALE O sequence.2 Approaching the start sequence.
The research team used mathematical modeling of protein binding and inhibition to study optimal dimer parameters. They decided to design a dimer with TALE A as the first TALE in the amino acid sequence and TALE O.2 Because the second dimer created a more repressive dimer than vice versa.
Finally, the researchers compared the inhibition of TALE dimers to LacI. The covalent TALE dimer showed comparable performance to LacI based on its modeling. “Here, we became very intrigued that we could artificially create something that could create DNA loops as strong as those seen in the lac repressor system,” Becker said.
“This is a very smart application of TALE,” he said. Adam BogdanovHe is a molecular plant pathologist at Cornell University but was not involved in the study. Bogdanove’s group was one of the first to write the sequence code for TALE. He said experimental and modeling work is an excellent approach to optimizing proteins and exploring their functions. “This is another powerful tool in the toolbox for adjusting or manipulating gene expression to understand gene function,” he said.
One interesting comparison going forward, Bogdanove said, is how efficiently TALE dimers suppress gene expression compared to CRISPR interference systems. Furthermore, improving the dimeric system so that the degree of inhibition can be controlled will help expand its applications, he said.
Becker and his colleagues are also interested in finding ways to make the system tunable and working with eukaryotic models. Unlike LacI, TALE proteins can be made to recognize arbitrary sequences. She explained that their team plans to test this TALE dimer suppression mechanism on new regions of bacterial and eukaryotic DNA using the parameters they identified.
