Robertson obtained the ligase ribozyme through a kind of test-tube evolution when he was a graduate student at the University of Texas, Austin, working in the lab of biochemist Andrew Ellington. Starting with a mixture of randomly synthesized RNA molecules and selecting for the desired properties, researchers are able to evolve RNA enzymes from scratch. In the Ellington lab, Robertson evolved the ligase ribozyme (called the L1 ligase) and determined which parts were critical for its function and which parts could be removed to create a "minimal construct."
At UC Santa Cruz, he began trying to grow crystals of the ribozyme so that he could use x-ray crystallography to determine its structure. Crystallizing RNA molecules is extremely difficult, and Robertson tried dozens of different versions of the ribozyme under different conditions before he succeeded. Using x-ray crystallography--which involves shining a beam of x-rays through the crystals and analyzing the resulting diffraction patterns--Robertson and Scott were then able to determine the three-dimensional structure of the ribozyme.
The ribozyme has three stems that radiate from a central hub. The active site where ligation occurs is located on one stem, and the structure shows that the molecule folds in such a way that parts of another stem are positioned over the ligation site, forming a pocket where the reaction takes place. A magnesium ion bound to one stem and positioned in the pocket plays an important role in the reaction, Robertson said.
The structure indicates that this artificially selected ribozyme uses reaction mechanisms that are much like those used by naturally occuring enzymes, Robertson said.
"The L1 ligase appears to use strategies of transition-state stabilization and acid-base catalysis similar to those that exist for natural ribozymes and protein enzymes," he said.
Source:University of California - Santa Cruz