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November 01, 2001
Evidence that a Ribozyme Evolved Multiple Times
Laboratory experiments designed to evolve new catalytic RNA
molecules, called ribozymes, have demonstrated that a type of
self-cleaving ribozyme found in highly divergent organisms might have
evolved independently multiple times.
Thomas R.
Cech, who is currently president of the Howard Hughes Medical
Institute (HHMI), and colleagues at the University of Colorado,
Boulder, discovered RNA catalysis in the early 1980s. Prior to Cech's
research on RNA, many scientists believed that proteins were the only
catalysts in living cells. A series of experiments done independently
by Cech and Sidney Altman at Yale University ultimately revealed that
RNA could also act as a biologic catalyst, a ribozyme.
“Since the hammerhead is a very simple motif, if any structure was going to arise independently multiple times, it would be something like this.”
Jack W. Szostak
The evidence for multiple origins of the hammerhead ribozyme, which
is found in organisms as diverse as plant viruses, newts, schistosomes
and cave crickets, was published in the November 1, 2001, issue of the
journal Nature by HHMI investigator Jack W.
Szostak and Kourosh Salehi-Ashtiani at Massachusetts General
Hospital.
According to Szostak, the evidence that the hammerhead ribozyme
might have had multiple origins arose from experiments in which the
scientists were attempting to create a faster self-cleaving ribozyme
for use in research. "There were two alternatives that had been
proposed for the evolution of this ribozyme," said Szostak. "Since the
hammerhead is a very simple motif, if any structure was going to arise
independently multiple times, it would be something like this," he
said. "But there was always the possibility that all these enzymes
descended from a single ancestral progenitor."
According to Szostak, one way to explore the origin of the ribozyme
is to use in vitro directed evolution, a technique pioneered by
Szostak to develop and isolate molecules with specific functions. In
vitro directed evolution involves generating large numbers of DNA
molecules with different sequences and imposing a selective pressure on
the mixture. Alternately, the scientists can impose selective pressure
on RNA or protein enzymes, to winnow out those with desired properties.
Then, they can work backward to determine the original DNA sequences
from which the selected molecules were derived.
In "evolving" self-cleaving ribozymes, Szostak and Salehi-Ashtiani
first generated a large random pool of DNA molecules that would code
for random-sequence RNAs. To prevent self-cleavage in this large pool
of ribozymes, they added a short piece of DNA to the cleavage site on
the RNA, thus protecting this site from the action of the ribozyme. The
scientists then started the reaction by adding magnesium ions and
"pushed" the selection for faster and faster cleavage by allowing less
and less time for cleavage in subsequent rounds of selection. They
purified those RNAs that had undergone self-cleavage. Next, they
reverse-transcribed the original full-length, self-cleaving RNAs back
into DNAs, replicated the DNAs, made large numbers of copies, and
repeated the process of inducing the new batch of RNAs to self-cleave.
To reveal the structures that had evolved, the researchers sequenced
RNAs from sixteen successive rounds of selection.
"We were quite surprised when we found that the selection was
completely dominated by hammerhead motif when we got up to the activity
levels found in biological molecules," said Szostak. The evolution of
the hammerhead motif likely resulted from the design of their
experiments, he said. "While a number of other laboratories have done
such selection experiments, they hadn't pushed as hard on the
activities, so most of the published results had shown lower-activity
ribozymes," he said.
"Clearly, once we required a certain level of activity, almost all
the sequences we saw emerge from our selection process were consistent
with formation of a hammerhead ribozyme," said Szostak.
Commenting on the studies, HHMI investigator Jennifer
A. Doudna, who studies RNA structure and catalysis at Yale
University, said, “It’s a very interesting finding, and not
necessarily one that would have been expected. It’s particularly
interesting that they set out initially to do a very different kind of
experiment to try to identify fast self-cleaving sequences. And while
one might have thought they would find something perhaps completely
different than any of the ribozymes found to occur biologically, the
surprising result is that the hammerhead is the best there
is.”
Szostak’s and Salehi-Ashtiani’s selection experiments
also yielded some unusual RNA ribozyme sequences, in which structural
elements expected to be conserved as necessary for self-cleaving
functioning were altered. Closer study of those aberrant RNA structures
might reveal insight into the self-cleaving mechanism of the hammerhead
ribozyme.
According to Doudna, basic understanding of the hammerhead
ribozyme’s catalytic mechanism has been lacking. “The
hammerhead was the first ribozyme structure determined by x-ray
crystallography,” said Doudna. “Since then, there has been
some very elegant work – by people such as Bill Scott of the
University of California, Santa Cruz, Olke Uhlenbeck at the University
of Colorado, Boulder, and Dan Herschlag at Stanford – to address
the mechanism of catalysis. But the frustrating thing is that, despite
all that work, it’s been very hard to nail that mechanism down
exactly. As ‘simple’ as this ribozyme would appear,
it’s actually quite complex,” she said.
Szostak and his colleagues plan to continue their efforts to achieve
faster self-cleaving ribozymes, which might be useful for in vivo
studies of cell function as well as therapeutics. Some ribozyme-based
therapies seek to use hammerhead ribozymes to block the production of
an unwanted protein in a metabolic disease by cleaving its messenger
RNA before the protein can be produced. However, said Szostak,
ribozymes have not proven active enough to be clinically useful.
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