While CRISPR may be the most notable genetic technology to emerge from microbes, many of the tools researchers use to control genes and their products are borrowed from bacterial systems. One of these is the lac operon, which regulates the expression of genes involved in lactose metabolism in Escherichia coli. Applications from lab benches to industrial vats use this to selectively turn on gene expression.
However, in most cases, researchers have only borrowed part of the lac operon system. They use the repressor protein, LacI, which physically binds DNA upstream of the gene promoter to block RNA polymerase, then add an inducer molecule that will remove LacI when researchers want to turn gene expression on. While this engineered system regulates the expression of genes, it requires high concentrations of LacI to consistently repress them.1 In comparison, the full form of the operon that occurs in E. coli includes additional binding sites for LacI that form a DNA loop, more efficiently repressing the transcription of the lac operon genes using less repressor protein.
One team is interested in leveraging this feature to develop a new approach to gene regulation. In a study published in Nucleic Acids Research, researchers at the Mayo Clinic created a novel protein that mimicked the dual-binding ability of LacI, offering a new approach to regulating gene expression in the future.2
LacI is a tetrameric protein, using two dimers to bind two specific, identical DNA sequences. This specificity narrows the ability to use the LacI system in cases where researchers canâ€t insert this sequence, prompting a search for a more flexible repressor protein that can recognize more regions of DNA.
“It was probably in about 2015 that we started working on the idea of more of a designer gene repression loop,†said Nicole Becker, a molecular biologist at the Mayo Clinic and study coauthor.
TALE proteins, short for transcription activator-like effector (TALE), are a class of proteins derived from plant pathogens in the Xanthomonas genus.3 TALEs recognize DNA using a repetitive series of 34 amino acid regions in which the 12th and 13th amino acid determines the nucleic acid that each segment will bind. Pathogens use these proteins to activate gene expression in plants that will promote the bacteriaâ€s survival, but scientists cracked this DNA-binding code to create custom TALEs that can recognize any sequence they want to target.4,5
In the present study, the team copied the double-headed nature of LacI by linking together two different TALE dimers, termed A and O2, that recognize distinct DNA sequences. They inserted these two sequences upstream of a reporter gene and used a colorimetric assay to determine whether the proteins repressed the reporterâ€s expression based on an absence of color. Their goal was to determine the parameters needed to achieve gene repression comparable to LacI.
First, the team assessed the effect that the order of the two TALE dimers in the protein had on repression efficiency. Previous research indicated that placing the stronger repressor farther away from the promoter improved repression, so they tested the degree of repression of A and O2.6 Then, they inserted the sequence for the stronger TALE A at their promoter distal site and the weaker TALE O2 closer to the start sequence.
The team studied optimum dimer parameters using mathematical modeling of protein binding and repression. They determined that designing the dimer with TALE A as the first TALE in the amino acid sequence and TALE O2 as the second created a more repressive dimer than the reverse.
Finally, the team compared the repression of their TALE dimer with LacI. The covalent TALE dimer performed comparably to LacI based on their modeling. “That was really where it became intriguing that we could artificially create something that could create a DNA loop that was as strong as that could be seen in the lac repressor system,†Becker said.
“It’s a very clever application of TALEs,†said Adam Bogdanove, a molecular plant pathologist at Cornell University who was not involved with the study. Bogdanoveâ€s group was one of the teams that originally described the sequence code of TALEs. He said that the experimental and modeling work was a good approach to optimize the proteins and explore their functions. “It’s another powerful tool in the toolbox for moderating or manipulating gene expression in order to understand gene function,†he said.
One comparison that Bogdanove said would be interesting to see in the future was how efficiently the TALE dimers repress gene expression compared to CRISPR interference systems. Additionally, he said that improving the dimer system so that the degree of repression could be regulated would help expand its application.
Becker and her colleagues are also interested in exploring ways to make the system tunable as well as studying it in eukaryotic models. Unlike LacI, TALE proteins can be made to recognize any sequence. She explained that their team will use the parameters that they identified to test this TALE dimer repression mechanism against new regions of DNA in bacteria and eukaryotes.
