Yale Researchers Solve Three-Dimensional Structure Of RNA Enzyme that Plays Key Role in Hepatitis Virus Replication

The discovery nearly two decades ago 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 stunning 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. Ferre-D’Amare 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, Doudna said.

“It is known that this RNA enzyme is essential for replication of the virus,” Doudna said. “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. Life as we know it cannot exist without DNA as the storehouse of genetic code, RNA as the genetic messenger, and proteins to carry out the chemistry of reproduction. Each requires the other two.

Since the discovery of RNA enzymes, however, many scientists have focused on RNA is the strongest candidate for 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,” said Doudna, who is a Howard Hughes Medical Institute assistant investigator at Yale.

Besides elucidating how life might have evolved, 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 said.

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,” she explained.

“Each one of those pieces makes a new virus, a process that is very efficient, very unexpected,” Doudna said. Furthermore, the enzyme accomplishes this feat faster than any other naturally occurring, self-cleaving RNA enzyme discovered thus far, with a cleavage rate of more than once per second.

“The virus is very smart to take advantage of an RNA enzyme to do the cutting and splicing without going to the bother of creating a protein to do it,” Doudna said.

The three-dimensional X-ray crystallography images created at Yale reveal a heart-shaped molecule with the chemically active site buried at the center. Resolution of the 72-nucleotide sequence is precise to 2.3 Angstroms, which is the width of about three atoms.

Now that the structural database for RNA enzymes is rapidly expanding, the prospects look brighter for eventually predicting an enzyme’s three-dimensional structure from its sequence of chemical building blocks, without requiring complicated imaging methods.

RIt would be an important accomplishment to solve an RNA structure simply by knowing its genetically specified sequence of nucleotides,” Doudna said. “If we had that kind of understanding of how atoms arrange themselves in three dimensions, 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.

Yale University is one of the leading centers in the world in the use of molecular imaging techniques to reveal the structure of key proteins and the complex interactions of proteins with both DNA and RNA.