Finally, Steven A. Jacobs, a Damon Runyon Cancer Research Foundation postdoctoral fellow working in Cech's lab, took a new tack. He reported his novel method and important findings in February in Nature Structural & Molecular Biology. Rather than trying to force the entire protein to crystallize, Jacobs used genetic engineering methods to create more than 10,000 different random TERT protein fragments, each produced by a different population of Escherichia coli bacteria. He genetically fused each fragment to green fluorescent protein (GFP), which glows when illuminated with ultraviolet (UV) light. The GFP would serve as a biochemical beacon, alerting Jacobs of any fragments that happened to fold correctly.

As expected, practically all the random fragments misfolded and formed insoluble inclusion bodies. “The inclusion bodies pulled the GFP inside them so that the proteins couldn't fluoresce nearly as brightly as they could if they were soluble—the GFP essentially goes down with the ship,” Cech explains. But some of the protein fragments formed structures that could fold into their correct shape as they do in the full-length protein, and they did remain soluble within the E. coli cells that produced them. “You just have to shine a UV lamp on the Petri dish and literally pick the bright green colonies off the plate with a toothpick,” he explains.

Jacobs purified the TERT-GFP fragments from several brightly glowing E. coli colonies, and sure enough, one of them crystallized readily, allowing the precise atomic structure of the protein to be determined by x-ray diffraction.

That fragment was from a region of the TERT protein chain called the N terminus. By comparing the sequences of the same region of TERT from several different species, identifying the common amino acid positions shared among all the species, and mapping those positions to their structural model, Jacobs and his colleagues inferred that a grooved cleft running through the protein must be important to the enzyme's function. After changing several of those key amino acids in the full-length TERT protein, they found that the enzyme lost its ability to assemble telomeres. “So we knew that this domain was necessary for function,” Cech says. They dubbed the fragment the “TEN domain,” for telomerase essential N terminus.

Further experiments showed that the TEN domain clamps onto the DNA near the end of the chromosome, positioning telomerase to begin building telomeres. “It's a part of the protein that is miles away from the active site [the part that catalyzes the chemical reaction],” Cech says, "yet it's completely essential for activity.” The hope, he says, is that scientists can now develop drugs targeting the TEN domain, preventing it from clamping to the chromosomes in cancer cells, and thereby nixing their immortality.

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