Yale Researchers Accidentally Discover Fluoride Genetic Response Mechanism

Structure model of a truncated fluoride riboswitch. Courtesy of Professor Breaker.

Yale Professor of Molecular Biophysics and Biochemistry Ronald Breaker and his group have recently determined the function of a previously unknown pervasive fluoride riboswitch, which now holds exciting potential for a variety of biomedical applications.

Riboswitches are portions of mRNA that bind to ligands to induce conformational changes that influence gene expression. Significant research is being conducted on riboswitches and their control mechanisms to shed light on the means of genetic regulation, and particular attention is being focused on the discovery of genes that each riboswitch regulates. Despite these research efforts, however, about one-third of all known riboswitches have not been matched to genes, and they are aptly dubbed as “orphans.” One such orphan, known as crcB, was given special attention because it is the second most distributed riboswitch among the three domains of life — and this is the riboswitch Breaker had identified.

The first step in understanding the function of this riboswitch was to find the ligand that activates it. For example, the guanine riboswitch controls guanine supply. The riboswitch maintains a conformation that allows transcription at low guanine levels. At higher concentrations, guanine binds to the riboswitch, causing it to fold and prevent transcription. Since there are many genes that are associated with the crcB orphan, it was difficult to determine a ligand for this riboswitch candidate. The only other riboswitch class associated with such a diverse group of genes responds to short RNA signaling compounds. Thus, these signaling compounds became the Breaker Lab’s first ligand candidate for experimentation. Using a technique known as in-line probing, the Breaker Lab assessed whether the RNA underwent a conformational change that is characteristic of riboswitch activation.

B. subtilis bacterial cells were transformed with a plasmid carrying the fluoride riboswitch fused to lacZ, and this gene is increasingly activated (blue) when fluoride is added. Courtesy of Professor Breaker.

After testing different compounds, the lab finally discovered a synthetic RNA ligand to which the riboswitch responded. To validate the findings, the team used the same ligand from a different chemical supply company; however, the expected response did not occur. Breaker and associates then tested various similar synthetic ligands from the original supplier, which, surprisingly, led to a conformational change. To investigate such a discrepancy, they contacted the ligand supplier for a list of possible contaminants. One of these contaminants was fluoride.

Because of the large accumulation of negative charges associated with RNA phosphate groups, lab members were pessimistic about the potential of the fluoride anion as a ligand. That didn’t stop chemistry graduate student Jenny Baker from going ahead and testing it. Contrary to expectations, the riboswitch responded to fluoride and the characterisitc conformational change was observed. The crcB orphan is now known as the fluoride riboswitch. “If we hadn’t ordered the sample from a company that didn’t purify the product properly, this could have remained a mystery for decades,” said Breaker.

Since the discovery, the Breaker Lab has expanded its scope to determine the functions of the genes regulated by the fluoride riboswitch, as dozens of genes elicit different responses to fluoride in different bacteria. Manipulation of the riboswitch or its associated proteins may set the foundation to trick cells into taking up fluoride or making fluoride more toxic, potentially leading to the discovery of antibiotics or antifungal agents.

Distribution of fluoride riboswitches and fluoride transporters in the three domains of life. Courtesy of Professor Breaker