Digging for RNA fossils

Ferre-D'Amare team discovers 'convergent evolution' of RNA and protein enzymes by solving the 3-D structure of the hairpin ribozyme
Dr. Adrian Ferre-D'Amare
Holding a model of protein, Dr. Adrian Ferre-D'Amare of the Basic Sciences Division displays the x-ray crystallography machine that has allowed him to solve the three-dimensional structure of an RNA enzyme. photo by Michelle Hruby

Whether you're cooking in a gourmet kitchen stocked with unlimited gadgets or making do with a wooden spoon and a frying pan, there aren't too many ways to scramble eggs.

The same can be true in biology, observes Dr. Adrian Ferre-D'Amare, an investigator in the Basic Sciences Division.

"Sometimes there are only one or two ways to carry out a particular chemical reaction - even when the enzymes doing the job are completely unrelated," he said.

By solving the three-dimensional structure of a ribozyme - a type of enzyme made of ribonucleic acid (RNA) instead of the typical protein - Ferre-D'Amare and postdoctoral fellow Dr. Peter Rupert learned that different types of molecules can evolve the same solutions to chemical problems.

First active structure

Their work, published last week in Nature, is the first to show the structure of a ribozyme in its active form.

Ribozymes are intricately folded RNA molecules that carry out enzymatic reactions once thought to be the sole domain of proteins. The hairpin ribozyme studied by Ferre-D'Amare and a protein enzyme called ribonuclease A carry out identical reactions - both cleave RNA, an information-containing molecule similar to DNA, into smaller pieces. Ferre-D'Amare and Rupert discovered that the hairpin ribozyme, found in a virus that infects tobacco plants, twists into an active form similar to the well-characterized ribonuclease A protein enzyme, whose structure was determined more than 30 years ago.

Biggest surprise

"This was probably the biggest surprise of our study," Ferre-D'Amare said. "Both enzymes have to twist their RNA substrate in the same fashion to cleave it. Because the two enzymes are completely distinct, we'd say this is an example of convergent evolution. Each independently evolved the same strategy to do the job, probably because there are limited ways to carry out this kind of chemistry."

Ferre-D'Amare's fascination with RNA stems in part from the theory that RNA may be the most ancient biological molecule.

"Cells need the ability to both store genetic information and to carry out catalysis," he said. "Modern cells use DNA for information andproteins for performing cellular chemistry, but the primordial cell is thought to have used RNA for both processes. The ribozymes we study today could be considered RNA fossils."

A crucial role for catalytic RNA was discovered last year when scientists solved the 3-D structure of the ribosome, a massive molecule made of both protein and RNA whose job is to decode a cell's DNA information into protein.

A research team at Yale University discovered that the ribosome's active site - where the actual protein synthesis takes place - is made of RNA, demonstrating that RNA catalyzes the chemical reaction required to make protein.

Since the ribosome is so large, making it relatively hard to study, Ferre-D'Amare focuses on understanding the chemical and physical properties of small catalytic RNAs.

Little-known 3D structures

Although the study of catalytic RNA is about 20 years old, little is known about how they are assembled into three-dimensional structures, Rupert said.

"Thousands of protein structures have been solved, but only a handful of RNA structures have been determined," he said.

Molecular structures are solved using a technique called X-ray crystallography, in which X-rays are beamed at crystals of the molecule, forming a distinct pattern as the rays are diffracted. Computer programs translate the diffraction patterns to reveal the three dimensional properties of a molecule.

In some cases, scientists can determine the resolution of the three-dimensional structure at the level of about a millionth of an inch.

To form the crystals of the hairpin ribozyme, Rupert utilized a trick that Ferre-D'Amare had developed while a postdoc at Yale: attaching a small portion of a protein, which helps the RNA crystallize more readily.

For the initial diffraction analysis, the crystal sample was subjected to X-rays in the Hutch's X-ray crystallography facility.

Use of synchroton
Once they were sure that their crystals were suitable for analysis, Rupert and Ferre-D'Amare used the Advanced Light Source synchroton at the Lawrence Berkeley National Laboratory to complete their diffraction studies.

The Hutch provides funding for membership in a research consortium that enables investigators to utilize the Berkeley synchrotron, a facility that accelerates particles close to the speed of light, for structural biology studies.

A synchrotron provides a more intense diffraction pattern than standard X-ray diffraction, providing improved resolution of the three-dimensional structure.

The hairpin ribozyme structure uncovered some longstanding questions about how RNAs catalyze reactions, Ferre-D'Amare said.

"Proteins are assembled from 20 different kinds of building blocks, called amino acids, which give proteins an enormous versatility in their catalytic ability," he said. "In contrast, RNAs are built from only four kinds of building blocks, called nucleotides, meaning that ribozymes must somehow make the most of what they've got."

The team found that the folding of the RNA into its active structure actually changes the chemical properties of its nucleotide building blocks, giving them a broader range of chemical capabilities needed for catalysis, Ferre-D'Amare said.

"We found that parts of the molecule change a lot as they come together to form the active site," he said. Ferre-D'Amare speculated that improved understanding of RNA active sites will make them attractive targets for drug design, since it is already known that certain antibiotics target the RNA component of the ribosome.

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