By Sean Henahan, Access Excellence

New Haven, CT (10/19/96)- New X-ray crystallography "snapshots" of ribozymes will aid research ranging from the search for the origins of life to the latest genetic engineering efforts, report Yale researchers.

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Ribozymes are specialized ribonucleic acid (RNA) molecules with enzyme-like properties. Their discovery earned Dr. Thomas Cech (Howard Hughes Medical Institute/University of Colorado) and Yale University biochemist Sidney Altman the 1989 Nobel Prize in chemistry. The discovery of ribozymes overturned the DNA-dogma that only proteins can trigger cell activity and helped clarify the "DNA/RNA-chicken or egg" questions about life's origins, since they are able to provide both the genetic code and the necessary activity for reproduction in one compact package.

Previously, most RNA molecules were thought to be passive genetic messengers responsible only for transcribing genetic code from DNA molecules and carrying that code to other sites in the cell for the production of proteins. The discovery that ribozymes could fold themselves directly into biologically active molecules following a self-contained genetic blueprint shook the world of biology.

"This capability to serve as a catalyst makes ribozymes a good candidate for being the first method of genetic reproduction and may provide the missing link in our understanding of how the earliest life forms could have evolved," says Dr. Yale biochemist Jennifer Doudna, assistant professor of molecular biophysics and biochemistry.

Recent Yale research by Dr. Doudna and colleagues yielded images showing how the ribozymes fold into a complex molecules capable of triggering cell activity. The new X-ray crystallography snapshots capture one-half of a self-splicing ribozyme molecule, revealing a compact, hairpin-shaped structure that is secured by two chemical clamps. The experiments were performed on ribozymes that cut and splice RNA in single-cell "Tetrahymena".

The discovery could help scientists design new drugs to fight lethal viruses, including the AIDS virus, and repair genetic errors that cause diseases ranging from cystic fibrosis to muscular dystrophy and sickle cell anemia. Ribozymes of the type depicted in this new research are already being developed to function as precision scissors that snip out flawed genetic segments from other RNA molecules and splice in corrected versions. The RNA scissors also can cut a virus's genetic code to shreds so it can't replicate.

The self-splicing ribozyme is by far the largest RNA molecule to have part of its 3-D chemical structure solved by X-ray crystallography in atomic detail. The resulting images provide insights into why an RNA molecule can arrange itself into a biologically active molecule while DNA apparently cannot.

The images also have enough detail to help guide genetic engineering, Professor Doudna said, and show the 3-D composition of a recurring motif in all ribozymes -- a chemical unit that makes up one blade of the scissors that snip genetic code. Resolution of the images is precise to 2.8 Angstroms, which is the width of one water molecule, or about three atoms.

"We found that this RNA molecule, which has about 9,500 atoms, contains two regions of contact that hold the two halves of the molecular structure together," Professor Doudna said.

"We also found that numerous metal ions-- specifically magnesium ions -- provide a scaffolding that stabilizes the structure. RNA also has a functional chemical group called a two-prime hydroxyl that can make numerous contacts to provide stability. That is one of the keys to why RNA can fold while DNA cannot."

According to Francois Michel and Eric Westhof, French biochemists, commenting in the journal Science, the images are "teeming with exciting detail.....Now that the structural database for RNA is rapidly expanding, the prospects look brighter for eventually predicting RNA three-dimensional structure from its sequence" of chemical building blocks, without requiring complicated imaging methods.

"It would be an important accomplishment to solve an RNA structure simply by knowing its genetically specified sequence of nucleotides," Professor 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."

BACKGROUND (Courtesy Yale University)

1. E. coli bacteria are turned into "factories" for producing large amounts of rare proteins. The technique, called cloning, involves inserting a specific gene segment, which encodes the protein, into a single bacterium. Millions of identical copies are made as the bacteria reproduce.

2. The cloned protein is purified and then crystallized. During the next step -- crystallography -- an X-ray beam passes through the crystals and is diffracted onto a detector, which records X-ray intensities as the crystal is rotated into many different orientations. These recordings are combined to produce a three-dimensional representation.

3. A cluster of Unix workstations serves the function of a lens to generate numerical data from which an image can be created. The data contain information about the locations of electrons in the molecule that can be used to calculate an "electron density map."

4. Advanced Unix computer servers convert the numerical density map into a visual 3-D representation, Using Silicon Graphics 3-D workstations, laboratory researchers fit a "backbone" through the electron locations to show how atoms are arranged in the molecule.