Breaker PHOTO BY MICHAEL MARSLAND/YALE UNIVERSITY

RNA—long thought to be just the middleman between DNA, the source of genetic instruction, and proteins, the workhorses of the cell--has gotten a makeover in the past few years. Now we know that RNA plays key roles in protein synthesis and genetic control. And thanks to Ronald R. Breaker, a professor of molecular, cellular, and developmental biology at Yale University, we also know that cellular RNAs sense environmental signals and regulate gene expression in response. Although it had been speculated as early as the 1960s that this might be the case, until recently biologists widely believed that such biosensing was a responsibility that cells left exclusively to proteins. Here, Breaker recalls his "Aha!" moment when he proved that cells use RNA, too. And he describes how that moment led to his lab's serendipitous discovery of a new kind of natural RNA-based enzyme (C&EN, March 22, page 6):

"A lot of the ideas we've pursued have been inspired by the theory that there was a time in biology, some 3 billion or 4 billion years ago, before the emergence of DNA or proteins, when RNA called all the shots. If there was once such an 'RNA world,' we speculated that one must be able to create RNA switches to sense the status of metabolic processes and click things on and off.

"Like everyone else, we assumed such RNA switches would long be extinct. But we thought they would make great biosensors, so we began to evolve 'riboswitches'--RNA molecules that can precisely recognize a specific metabolite, and either change shape or catalyze an enzymatic reaction in response--in the test tube. By the late 1990s, it became clear that we could build these riboswitches fairly easily, that we could build lots of different ones, and that they performed so well that you had to wonder why nature wasn't using them.

"One summer morning in 2000, the lab all got together for group meeting. Halfway through I stopped the meeting and said, 'OK, we can make these things very readily in the test tube. They work very well. They must be in modern cells.' I made two bets with my students: One, if there were riboswitches in modern cells, they would be located within messenger RNAs where they could control protein production. And two, biologists have already encountered riboswitches--they just didn't know it. Everyone was expecting that genetic factors that sense metabolites would have to be proteins, so when they described their work, it would go something like this: A research group identifies a gene whose expression is controlled by metabolite X. When metabolite X levels rise, the gene is shut off. But the researchers just can't find the protein that binds X and shuts down the gene.

"The very next day, a postdoc in my group came to me with a paper he had dug up in the library describing exactly such a mystery. We set about trying to figure out whether the mysterious protein biosensor for coenzyme B-12 could in fact be a riboswitch.

"The experiment we did relies on the basic chemical principles of RNA as a polymer. Unstructured RNA spontaneously breaks apart, but highly structured RNA falls apart very slowly. That's because the sugar-phosphate internucleotide linkages of RNA can undergo nucleophilic attack: The sugar's 2' hydroxyl attacks the phosphorus center of the linkage. But the reaction will only occur if the phosphate and the hydroxyl group are in the right stereochemical orientation. In a floppy RNA, the nucleotide linkages are allowed to sample all kinds of different geometries--and when it finds the right geometry, the RNA falls apart. But the nucleotide linkages in an RNA that's highly structured rarely sample the required geometry.

"The putative riboswitch must change shape when it binds coenzyme B-12, so we looked at the putative riboswitch's pattern of spontaneous cleavage with and without coenzyme B-12. As we suspected, the RNA fell apart in different places in the presence and absence of coenzyme B-12—and only coenzyme B-12. Close analogs of the metabolite didn't show the same effect. By late 2001, we were convinced that modern cells were indeed using riboswitches to regulate genes in response to environmental cues.

"This was the tipping point that made the field stop speculating about riboswitches and start embracing them. We went on to investigate five more similar mysteries in the literature and found a riboswitch was behind each one. But we figured there were likely many more out there—so we developed a bioinformatics tool to search microbes' genomes for similar riboswitches.

"That search turned up a particularly promising stretch of RNA that had all the structural hallmarks of a riboswitch. It was always present upstream of the coding region for an amidotransferase enzyme that produced the sugar glucosamine-6-phosphate, a key metabolite in cell-wall biosynthesis.

"In the last days of September 2003, we did the same experiments as before to prove that it was indeed a riboswitch. But when we let the RNA incubate for a short time it was completely destroyed—it completely cut itself in one position. Spontaneous cleavage events that are a reflection of the RNA's structure should be sprinkled throughout the RNA, and all of them should cleave at roughly the same speed. But one particular spot in the RNA was being cleaved at a speed that's 10 to 100 times as fast as the best spontaneous cleavage. It had to be due to the action of a catalyst.

"We figured the RNA had an interesting, complex structure that was accelerating self-cleavage at that particular site. The chemistry screamed that we had no other choice but to declare this thing a ribozyme--an RNA-based enzyme, of which there are only eight known classes found in nature.

"Still, our new ribozyme's activity was too slow to be biologically relevant. But when we tested this system with glucosamine-6-phosphate, the activity took off. With the sugar, the ribozyme became 1,000 times faster at cleaving at the hot spot than without it. In the presence of glucosamine-6-phosphate, cleavage becomes fast enough to be of biological relevance, and we knew that we had found a new kind of ribozyme, one that plays a fundamental role in cellular metabolism.

"For me, the most satisfying aspect of this research is that fundamental chemical principles of RNA and solid hypotheses about what RNA could do eventually led us to riboswitches and ultimately to this ribozyme."

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