|
"Which came first, nucleic acids or proteins? This
question once seemed an intractable paradox, but with the discovery of
ribozymes, it is now possible to imagine a prebiotic 'RNA World' in
which self-replicating ribozymes accomplished both tasks," said
William Scott, associate professor of chemistry and biochemistry at UC
Santa Cruz.
Scott and postdoctoral researcher Michael Robertson
determined the structure of a ribozyme that joins two RNA subunits
together in the same reaction that is carried out in biological
systems by the protein known as RNA polymerase. Their findings are
published in the March 16 issue of the journal Science.
"An RNA-dependent RNA polymerase ribozyme is the
foundation of the entire RNA World hypothesis," Robertson said. "With
that, you would have an RNA capable of making copies of itself;
mutations or errors in some copies would result in variations that
would be acted on by Darwinian natural selection, and the molecules
would evolve into bigger and better ribozymes. That's what makes this
structure so interesting."
Robertson and Scott determined the structure of a
ribozyme that is not an entirely self-replicating RNA molecule, but it
does carry out the fundamental reaction required of such a molecule--a
"ligase" reaction creating a bond between two RNA subunits.
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. |
