October 09, 1998
Ribozymes Come Ready for Action
A structural model of the ribozyme from the human pathogen hepatitis delta virus.
Using x-ray crystallography, two research teams from the Howard
Hughes Medical Institute (HHMI), working independently, have found that
catalytic RNA, like its protein cousins, is ready to function as soon
as it is produced.
Ever since HHMI investigator Thomas Cech at the University of
Colorado in Boulder uncovered the catalytic properties of RNA in 1982,
researchers have been diligently studying these ribozymes. Scientists
have since discovered more than 500 ribozymes in a diverse range of
organisms and have found that they share many similarities with their
more widespread protein cousins, enzymes.
"The finding of an enzyme-like active site fits right in with the
story we've been building about ribozymes. But a picture is worth a
thousand words and now we've got the picture," says Cech, who won the
1989 Nobel Prize in Chemistry for his discovery of ribozymes.
The results of Cech's group, spearheaded by HHMI associate Barbara
Golden, appear in the October 9, 1998, issue of Science. They
describe the three-dimensional structure of the largest ribozyme ever
crystallized, which comes from the organism Tetrahymena
thermophila. In the October 8, 1998, issue of Nature, HHMI
investigator Jennifer
Doudna and colleagues from Yale University detail the structure of
a smaller ribozyme from the human pathogen, hepatitis delta virus
(HDV).
Crystallizing RNAs is notoriously tricky. Crystals contain billions
of molecules that must be aligned precisely if they are to provide a
sharp image of a molecule's three-dimensional structure. But RNA
molecules are difficult to align because they don't usually have many
surface "nooks and crannies" to help the molecules stick together
within the crystal. While Doudna's group could easily crystallize the
viral ribozyme, most of the crystals they created didn't interact well
with x rays. Inspired by "desperation," Doudna's team came up with a
nifty trick for overcoming the problem.
Doudna's group attached an RNA-binding protein onto a region of the
viral ribozyme where it wouldn't interfere with the ribozyme's
activity. The protein provides surface contacts that enable the
ribozyme-protein complex to pack snugly into a crystal, allowing the
researchers to create a much higher resolution image than they could
have with the RNA alone. Using their new technique, Doudna's group
attained an image with a resolution of 2.3 angstroms, which is good
enough to see the exact position of every nucleotide in the
ribozyme.
Cech's group crystallized the Tetrahymena ribozyme without
help from proteins, but their molecules didn't pack as tightly as those
from HDV. The looser packing resulted in an image resolution of 5
angstroms, high enough to see the molecule's overall architecture while
leaving the picture "a little blurry around the edges," says Cech. He's
confident that future efforts will produce an image with greater
resolution.
 Despite the difference in resolution, both
groups produced images revealing active sites that are largely
preorganized—they don't require major shape changes in order to
catalyze reactions. The active sites of both the Tetrahymena
ribozyme and the viral ribozyme have tight binding pockets that sit
ready and waiting for the right RNA molecules to come along. This
preorganized structure wasn't seen in the hammerhead ribozyme, the only
other ribozyme crystallized previously. The hammerhead has an active
site that is more like a folding chair in that it must change shape
before it can do its job.
The latest x-ray pictures will help scientists in their search for
nature's rules for building RNA scaffolds. Doudna says that given RNA's
diversity, it's a sure bet that ribozymes use a variety of catalytic
strategies. She hopes future studies will uncover some of these
strategies. "Having structures available will allow us to see what
those active sites really look like and then we're able to go in and
really ask much more specific questions using other techniques," Doudna
says.
Doudna's work provides a new tool for creating RNA crystals, and
together with Cech's results, shows that crystallizing RNA is feasible
despite previous doubts that RNA might be too "floppy" to crystallize
well. "There are a lot of RNAs out there that are of biological
interest, and I think this will be an incentive for more people to work
on RNA crystallography," says Cech.
Illustration: (Top) Adrian Ferré-D'Amaré/Jennifer Doudna, HHMI at Yale University, (Bottom) Thomas Cech, HHMI at University of Colorado, Boulder.
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