August 11, 2000
High-Resolution Image Illuminates Catalytic Engine of the Ribosome
The image in the foreground shows the RNA/protein architecture of the large ribosomal subunit with the active site highlighted. The background shows a schematic diagram of the peptidyl transferase active site of the ribosome.
Using a high-energy x-ray beam to probe fragile crystals of RNA and
protein, researchers have obtained the most detailed images ever
produced of the cellular factory where amino acids are linked into
chainlike proteins.
The studies illuminate the basic structure of the ribosome, a
protein-making machine found in all cells. These insights include the
first unequivocal proof that the ribosome is a ribozyme, an RNA
enzyme.
In two articles published in the August 11, 2000, Science,
researchers led by Thomas A. Steitz a Howard Hughes Medical Institute
investigator at Yale University, report that they have obtained the
atomic structure of the 50S subunit of the ribosome at a resolution of
2.4 angströms. An angström is one ten-billionth
(10-10) of a meter.
The ribosome is a large molecular complex of RNA and protein. When
ribosomes are isolated from cell extracts, two different fractions are
obtained, representing two subunits. The smaller 30S subunit binds the
messenger RNA that constitutes the protein's genetic blueprint, as well
as the transfer RNA that carries each specific amino acid to be added
to the growing chainlike protein molecule. The larger 50S subunit
catalyzes the formation of the bond between each amino acid and the
growing protein chain.
Steitz and his colleagues at Yale University used the 2.5 billion
electron volt x-ray beam at Brookhaven National Laboratory's National
Synchrotron Light Source to perform x-ray crystallography on crystals
of 50S subunits that were produced with osmium and iridium atoms
attached to act as landmarks. Additional data were gathered using the
Advanced Photon Source at Argonne National Laboratory.
In x-ray crystallography, protein crystals are bombarded with
intense x-ray beams. As the x-rays pass through and bounce off of atoms
in the crystal, they leave a diffraction pattern, which can then be
analyzed to determine the three-dimensional shape of the protein.
"Our previous maps of the 50S subunit at nine- and
five-Ångström resolution gave us some hints at the
structure, but not until we achieved the 2.5-Ångström
resolution could we resolve the atomic structure of all 100,000 atoms
that are well ordered in the crystal," said Steitz. "This structure is
about four times larger than any other such structure that has ever
been determined, and the 3,000 nucleotides of RNA increased the amount
of known RNA structure by about 4 to 5 fold."
According to Steitz, the process of achieving such high resolution
meant painstakingly improving the process of growing larger, more
complete ribosome crystals, and solving structures of those crystals at
progressively higher resolution. Each lower-resolution map provided
information that could help the scientists understand the ultimate
high-resolution map, he said.
"I think we were amazed at each stage at the overwhelming complexity
of the RNA folding in the ribosome," said Steitz. "But I think the most
surprising observation was that the proteins were embedded among the
RNA helices, penetrating into the interior of the ribosome like
tentacles."
Such penetration of proteins explains why previous researchers had
not been able to show that the ribosome depended solely on RNA as its
catalytic molecule," said Steitz.
"Since (HHMI President) Thomas Cech had shown that RNA could have
catalytic activity, we had suspected that the 50S subunit was basically
a ribozyme," said Steitz. "However, there was no proof. Nobody had been
able to show that the RNA by itself showed catalytic properties in the
absence of the protein. Now we can see that part of the reason is
probably the nature of these proteins that are holding the ribosome
together.
"Our structure shows that these proteins are deeply embedded in the
RNA and are essential for its folding. And it shows unambiguously that
the ribosome is a ribozyme because we can see where the substrate binds
and there's no protein atom near enough to that site to produce any
catalytic activity."
The structure also provides intriguing insights into how the
ribosome might originally have evolved, perhaps as a machine to make
short proteins, or peptides, said Steitz.
"Earlier experiments by Cech and others had shown that it was
possible to create RNA molecules that have some of the catalytic
properties of the ribosome in peptide synthesis," he said. "Now we can
see in this structure that some aspects of the native ribosome reflect
some aspects of those RNA molecules produced through in vitro
evolution. So, the expectation that a small RNA molecule could have
evolved to catalyze peptide bond synthesis is not a far stretch.
"However, that peptide-making RNA molecule would not have been
directed by messages from some early genome," he added. "How evolution
managed to progress from making a random peptide to messenger-directed
synthesis, we haven't a clue."
According to Steitz, the latest high-resolution structure offers a
pathway to far deeper understanding of the protein-assembling
machinery. The researchers are planning further studies to understand
how the messenger RNA and components of the growing protein are
oriented in the ribosome's catalytic active site. They will also
explore how the ribosome structure influences the chemical properties
of the molecular groups in the active site. And, the scientists will
seek to understand how the multitude of magnesium and potassium ions
and water molecules integrate into the ribosome and stabilize it.
"We're certainly not done with the scientific challenges presented
by the ribosome," said Steitz. "Although I must say I do feel as if
we're standing on Mount Everest at the moment and I'm now looking to
find K2."
Image: Nenad Ban, Poul Nissen, Jeffrey Hansen, Peter B. Moore, Thomas A. Steitz
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