New Clues To Origin Of Life Could Benefit Genetic Engineering: 3-D Structure Of A Large Portion Of An RNA Enzyme Solved

September 20, 1996

3-D Structure of a Large Portion of an RNA Enzyme Solved Cynthia L. Atwood, Science Correspondent
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Embargoed by the journal Science until 5 p.m. Sept. 19, 1996, EDT

New Clues to Origin of Life Could Benefit Genetic Engineering: 3-D Structure of a Large Portion of an RNA Enzyme Solved

New Haven, CT - Which came first, the chicken or the egg? A possible solution to this ancient evolutionary riddle is found in a single class of molecules that appears to have functioned figuratively as both chicken and egg early in the evolution of life, perhaps even providing the first method for primitive cells to reproduce.

On the cover of the Sept. 20 issue of the journal Science, stunning "snapshots" reveal the three-dimensional structure of a large molecule in this chicken-and-egg category -- a specialized ribonucleic acid (RNA) molecule called an RNA enzyme, or ribozyme. The images show how the ribozyme folds itself into a complex molecule capable of triggering cell activity

The discovery, which was made by a team of scientists led by Yale biochemist Jennifer Doudna, 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. Collaborating on the research was Nobel laureate Thomas Cech of the Howard Hughes Medical Institute/University of Colorado.

Other members of the research team were graduate student Jamie H. Cate and research assistant Kaihong Zhou of Yale; and Anne R. Gooding, Elaine Podell, Barbara L. Golden and Craig E. Kundrot of the University of Colorado.

The discovery of ribozymes, which earned Dr. Cech and Yale University biochemist Sidney Altman the 1989 Nobel Prize in chemistry, is the basis for a whole new branch of genetic engineering. Ribozymes of the type depicted in this new research, for example, are 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.

One Compact Package for Genetic Code and Cell Division

Scientists had puzzled over which came first -- DNA genetic code or proteins. Without DNA, there would be no way to create proteins, which regulate all biological activity, including the building of bones, blood and muscles. But without proteins, there would be no activity to enable the cell to reproduce. Ribozymes, however, supply 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. It came as a surprise to learn that ribozymes, a type of RNA, can fold themselves directly into biologically active molecules following a self-contained genetic blueprint.

That discovery, made by Professors Altman and Cech in separate experiments performed in the late 1970's and early 1980's, forced the rewrite of many textbooks to remove the dogma that only proteins can trigger cell activity.

"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. Doudna, assistant professor of molecular biophysics and biochemistry.

The new X-ray crystallography snapshots, which capture one-half of a self-splicing ribozyme molecule, reveal a compact, hairpin-shaped structure that is secured by two chemical clamps. The experiments were performed on ribozymes that cut and splice RNA in Tetrahymena, which are single-cell, pond-dwelling creatures that Professor Cech used in his prize-winning research.

First Large Ribozyme Thus Analyzed

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 tantalizing 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 who reflect on the recent discovery in a "Perspectives" article published in the same issue of 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."

Note to Editors: Jennifer A. Doudna, who joined the Yale faculty in 1994, received her Ph.D. degree in biochemistry from Harvard, where she studied with Professor Jack Szostak, and was a post-doctoral fellow in Professor Thomas Cech's laboratory at the University of Colorado, Boulder. For interviews, contact her at (203) 432-3108. Contact Professor Cech at (303) 492-8606.

The Process of Mapping Molecules with X-ray Crystallography

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.

Scientists are using knowledge of molecular structure to create medications that mesh, like a key in a lock, with specific proteins, thus reducing unwanted side effects and making disease treatments more effective. They also are pinpointing and attempting to correct inherited diseases that are caused by defective or insufficient proteins, and are tailoring new proteins to improve the size and vigor of plants and livestock.

Researchers in Yale's molecular biophysics and biochemistry (MB&B) department specialize in the use of a technique called X-ray diffraction or crystallography -- a well-established procedure for determining three-dimensional molecular structure by probing crystals with an X-ray beam. The technique reveals the locations of individual atoms in gigantic molecules with molecular weights of as much as 100,000.

Here is the step-by-step process for X-ray crystallography:

* 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.

* 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.

* 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."

* 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.

Yale University

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