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The researchers used a machine called an "optical trap" to grab and
hold the ends of an RNA molecule with laser beams. Based on technology
developed by Bell Labs researchers in 1986, the machine was designed
by a team led by Steven Block, the Stanford W. Ascherman, M.D.,
Professor and a professor of applied physics and of biology. The
optical trap allows them to hold the ends of the RNA tightly, so they
can pull it pin-straight, then let it curl up again. In the Feb. 1
issue of Science, their paper, of which Block is senior author,
describes the development of every loop and fold in one particular RNA
riboswitch, and the energy it takes to form or straighten each one-an
unprecedented achievement that opens the door for equally thorough
studies of other molecules and their behaviors.
The researchers are the first to study the energy and folding behavior
of a riboswitch in this detailed, physical way. More important, they
are the first to use directly applied force to determine how a
molecule makes a three-dimensional bundle, a tertiary structure. No
other research has tracked the formation of such a complex structure,
fold by fold.
Previous studies typically have used biochemical techniques rather
than lasers, which can directly grab and tug the RNA. Biochemical
techniques give less clear estimates of how molecules fold in real
time. They often give a description of the molecule's average folding
behavior, which must be interpreted by mathematical models.
Crystallography-a technique involving freezing the molecule in
place-provides a good picture of its shape, but not how it forms or
the energy involved.
"What we're interested in is understanding, in a very fundamental way,
how biomolecules take the shapes they do, and how they perform the
functions they do," Block said. "No one has been able to explore in
great detail tertiary structure yet." RNA riboswitches must have this
tertiary structure to work.
"Most RNAs just make secondary [two-dimensional] structure. But the
ones that really do stuff," he added, "those all have tertiary
structure."
What RNA can do
RNA has the job of copying the genetic code from DNA (transcription),
and using that code to build the proteins organisms need to live (translation).
To make RNA, a protein called RNA polymerase moves along the length of
a strand of DNA. It reads a pattern in the building blocks of DNA,
nucleic acids whose names are abbreviated A, C, G and T, and it makes
RNA with a complementary pattern. This long strand of RNA is then the
recipe for a specific protein. Another structure called a "ribosome,"
which is also made of RNA, then reads this recipe and makes a protein
to order.
The RNA copied from DNA generally does not twist up very much, often
only forming two-dimensional loops or tight bends called "hairpins."
Occasionally, its loops and hairpins form a three-dimensional
structure that does nothing. Sometimes, though, this snarl of loops
and hairpins works as a riboswitch. The RNA begins to bundle up while
it is being made, so the jumbled portion is attached to a tail still
under construction. The riboswitch must have a tertiary structure,
because it likes to make a pocket and grab small molecules. When a
riboswitch clutches the right molecule, it folds up even more tightly,
tugging on its own incipient long tail and changing its shape in a way
that will affect its eventual protein product. That RNA tail usually
has a hairpin fold that straightens out when pulled. By tugging out
this kink in the RNA, a riboswitch changes how the RNA is translated
into protein, effectively turning the gene on or off.
The riboswitch Block's team studied grabbed onto a molecule called
adenine, the nucleic acid dubbed "A." Whenever the riboswitch gripped
a free-floating adenine, a gene that makes a protein crucial to
adenine production stopped working correctly. The RNA responsible for
translating it to the protein had changed shape. The riboswitch
regulated how much adenine was available in the cell; when there was
plenty, it shut down the adenine factory. Before scientists discovered
riboswitches, they thought only proteins controlled genes this way. "Your
average RNA at random is not going to do that," Block said. "These are
highly evolved things."
The closest look
The researchers who study molecular folding in Block's lab cannot
actually see an RNA molecule under the microscope, but they can see
two polystyrene beads; they attach one on either end, and that creates
a dumbbell shape the laser beams can manipulate. Their largest beads
are 1,000 nanometers across, so 1,000 of them lined up would be a
millimeter long. The beads are enormous relative to the RNA, and so
are the lasers holding them. To keep the lasers from coming too close
together and merging their light into a single beam, the researchers
need to attach some extra length to the RNA. To do this, they tack a
long strand of DNA on one side.
Under the microscope, the two plastic beads look like tiny pearls
against a gray backdrop. The researchers pull the beads apart, taking
into account two factors: force and extension. By understanding how
much force it takes to cause a certain amount of extension of the RNA,
they can describe with unsurpassed accuracy how the folds form and the
energy needed to make each fold happen.
"When you pull it apart, different structures will pop open-pop, pop,
pop-and you can see the order in which different structural elements
get pulled apart," Block said. "You can map out the order in which the
pieces come together, for both folding and unfolding."
Learning by force
To build a clear picture of how their riboswitch folded in real time,
the researchers mapped out the energy of the molecule's folding based
on the forces required to uncurl it and the time the RNA took to
re-curl. Block calls the energy graph the "crown jewel of the work,"
adding that "all the numbers you'd like to know about this folding
sequence are right in front of you in that diagram."
Block's team could only attain this detailed "energy landscape" of the
RNA's folding by physically toying with the molecule. The particular
RNA they studied folds four times, and each time it adopts a more
stable, more comfortable configuration with lower energy. If it grabs
an adenine, it hangs on tightly because it is in its most stable state.
But because molecules are always jiggling, sometimes a fold pops open
briefly. The more stable each fold is, the less likely it is to come
undone. The researchers stretched out the RNA to study all four folded
states, noting how stable each one was.
Using force, Block's team described not only the energy of each fold
in the RNA, but the energy it needed to go from one folded state to
the next, and how often the folds popped open and closed in real time.
The researchers watching little white beads move under the microscope
got the closest look yet at how a molecule with a three-dimensional
structure behaves in life, thanks to a pair of keen, green lasers and
a little judicious tugging. "It's so cool to be able to take a single
molecule and bend it to your will," Block said.
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