July 20, 2000
Technique Shows Ratcheting Motion of Ribosomes
Using a technique called three-dimensional cryo-electron microscopy,
researchers have detected a ratcheting rotation deep inside the cell's
tiny protein-making "factory" at a key point in the protein
construction process.
The ratcheting motion of the ribosome, a large protein-making
organelle, consists of a rapid rotation of one of the ribosomal
subunits relative to the other just as the messenger RNA (mRNA) and the
attached molecules of transfer RNA (tRNA) are advanced by one codon.
Howard Hughes Medical Institute investigator Joachim Frank and
colleague Rajendra Kumar Agrawal at Health Research Inc., at the
Wadsworth Center in Albany, New York, reported their discovery in the
July 20, 2000, issue of the journal Nature.
The finding highlights the burgeoning ability of researchers to
tease apart the details of protein synthesis—a key biological
process that occurs in every living cell. "This just gives us a flavor
of what is yet to come," said Frank of his laboratory's efforts to
understand the intricate contortions of the ribosome. "We have also
seen other movements that play a key role in ribosomal function and we
will continue to explore those."
The ribosome is a large molecular complex of RNA and protein. When
ribosomes are isolated from cell extracts, two different fractions are
obtained. One fraction consists of a smaller, 30S subunit, and the
other, a larger 50S subunit. The 30S subunit binds the mRNA, as well as
tRNA—which carries each specific amino acid to be added to the
growing chainlike protein molecule. As the 30S unit, thus, helps in
"reading" the mRNA, the larger 50S subunit catalyzes the formation of
the bond between each amino acid and the growing protein.
After each bond is made, a molecule called elongation factor G binds
to the ribosome. This binding, along with the chemical reaction of the
energy-containing molecule GTP, triggers the translocation, or
movement, of the mRNA and the tRNAs attached to it by one unit, or
codon. Once it is moved, the mRNA can be read to determine the next
amino acid to be added.
A central question, said Frank, was whether the two ribosomal
subunits underwent some sort of movement relative to one another to
facilitate the translocation.
"There have been hypotheses about subunit movement for years," said
Frank. "But there has never been a direct confirmation of this. The
problem was that all the evidence was indirect. Scattering studies, for
example, indicated changes in specific regions of subunits between one
state and the other, but they were never directly observed. And it is
only now, with cryo-electron microscopy, that we can visualize the
ribosome with such clarity."
Three-dimensional cryo-electron microscopy (cryo-EM) is one of the
few techniques capable of visualizing large, dynamic molecules. In
preparing for cryo-EM, researchers first immerse the ribosomes in water
solution and then abruptly freeze them in supercold liquid ethane. The
rapid freezing imprisons the ribosomes in ice, thus preserving their
native structure. Using an electron microscope with a low-intensity
beam to avoid damaging the molecules, scientists obtained images of the
thousands of captive ribosomes. The scientists employed sophisticated
computerized image analysis to produce a detailed, three-dimensional
map of ribosome motion from the otherwise low-contrast, noisy images
produced by the electron microscope.
To capture the ribosome in the act of moving mRNA and tRNA, the
scientists added a non-working analog of GTP along with elongation
factor G to the ribosomes, effectively stopping protein synthesis dead
in its tracks.
"Using this analog made it possible to capture a state in which the
elongation factor is bound to the ribosome, but there is no further
progress," said Frank. "So, the whole system is frozen by a chemical
means."
Their analysis of the chemically frozen ribosomes revealed that when
the elongation factor and GTP bind to the 30S subunit, it rotates about
six degrees with respect to the 50S subunit. And after the GTP chemical
reaction, the 30S subunit rotates back.
"This rotation goes along with other movements," said Frank. "If one
looks at the entry channel for the mRNA into the ribosome, it normally
appears to narrow and widen as the subunit moves back and forth. It is
exactly the expected opening if one thinks that in one state the mRNA
has to be free to move and in the other state it needs to be secured
and prevented from moving."
According to Frank, further advances in cryo-EM—together with
detailed atomic-resolution analysis from x-ray crystallography
studies—should provide even greater insight into ribosomal
movement during protein synthesis.
Frank and his colleagues are now developing a technique to
synchronize ribosomal processes before freezing, so that the thousands
of ribosomes that are frozen in a given preparation are halted at
precisely the same point in protein synthesis. Such synchrony would
enable the researchers to study the mass of ribosomes at any particular
point, to further understand how ribosomes aid in the construction of
proteins.
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