Discovery reveals how RNA enzyme replicates
hepatitis delta virus

Nearly two decades ago, the discovery of naturally occurring ribonucleic acid (RNA) enzymes earned Yale biochemist Sidney Altman and University of Colorado researcher Thomas Cech the 1989 Nobel Prize in Chemistry. In separate experiments, Altman and Cech exploded the myth that RNA is merely a passive carrier of genetic code by showing that it also can carry out chemical reactions necessary for cell growth.

Following up on that discovery, Yale biochemist Jennifer Doudna solved the three-dimensional chemical structure of a large part of the RNA enzyme first discovered by Cech. In 1996, she and Cech showed how this portion of the specialized enzyme folds itself into a complex molecule capable of triggering cell activity.

Now both Doudna and Cech have added to the growing storehouse of molecular information about RNA enzymes with X-ray crystallography images that provide a more precise picture of how they function. Their work brings the total number of RNA enzymes that have been fully visualized to just three, compared with thousands of protein enzyme structures now on file.

In the Oct. 8 issue of the journal Nature, Doudna and her colleagues (Yale postdoctoral associate Adrian R. Ferré-D'Amaré and research technician Kaihong Zhou) reveal the crystal structure of an RNA enzyme that plays a role in the replication of the hepatitis delta virus -- the only example of an RNA catalyst found thus far in a human pathogen. Hepatitis delta, a secondary infection that sometimes occurs in patients who have hepatitis B, is a problem primarily in developing countries, where it often is fatal, explains Doudna.

"It is known that this RNA enzyme is essential for replication of the virus," says Doudna, who is an associate professor of molecular biophysics and biochemistry. "Using our knowledge of its molecular structure, it may be possible to design pharmaceuticals that interfere with its function and stop the progression of the disease."

In the Oct. 9 issue of the journal Science, Cech reveals a low-resolution image of an RNA enzyme from Tetrahymena thermophila, the same single-cell, pond-dwelling creatures he used in his Nobel Prize-winning research. Images from both researchers show active catalytic sites located in crevices, much like those found in protein enzymes.

The discoveries provide important clues to the chicken-or-egg dilemma of which came first -- DNA, RNA or proteins. All three play important roles in life as we know it: DNA as the storehouse of genetic code, RNA as the genetic messenger, and proteins as the means of carrying out the chemistry of reproduction. Each requires the other two.

Since the discovery of RNA enzymes, however, many scientists now believe it is most likely that RNA was a precursor of all life forms, the single molecule that served as both chicken and egg some 4 billion years ago by providing genetic code as well as the first method for primitive cells to reproduce.

"In addition to RNA's dual function as genetic molecule and as enzyme, RNA serves important roles today in all living systems as the carrier of genetic instructions from DNA and as an orchestrator of all protein synthesis," says Doudna, who is also a Howard Hughes Medical Institute assistant investigator at Yale. RNA enzymes have shown great promise for clinical treatment, functioning as precision scissors that could snip out a flawed gene segment and splice in a corrected version. "This method has potential for treating diseases ranging from cystic fibrosis to muscular dystrophy and sickle cell anemia," Doudna says.

According to the Yale researchers, the hepatitis delta virus uses RNA scissors to cut its genetic material into viable segments during an infection. "As the virus is replicated in infected cells, the genome of the virus becomes a long spiral of RNA that has to be cut into pieces. The RNA at the end of the spiral folds itself into an enzyme and cuts the spiral into successive segments," Doudna explains.

"Each one of those pieces makes a new virus, a process that is very efficient, very unexpected," she says. Furthermore, the enzyme accomplishes this feat faster than any other naturally occurring, self-cleaving RNA enzyme discovered thus far.

Now that the structural database for RNA enzymes is rapidly expanding, scientists may eventually be able to predict an enzyme's three-dimensional structure from its sequence of chemical building blocks, without requiring complicated imaging methods, Doudna says. "If we had that kind of understanding ... it would not only speed drug design but also give us insights into how to fix genetic defects."

Funding for this research was from the National Institutes of Health, Howard Hughes Medical Institute, the Jane Coffin Childs Memorial Fund for Medical Research, the Searle Scholars program, the Beckman Young Investigator award and the David and Lucile Packard Foundation.