All
the crucial proteins in our bodies must fold into complex shapes to do
their jobs. These snarled molecules grip other molecules to move them
around, to speed up important chemical reactions or to grab onto our
genes, turning them “on” and “off” to affect which proteins our cells
make.
Recently, scientists have discovered that RNA-the stringy molecule that
translates our genetic code into protein-can act a lot like a protein
itself. RNA can form loopy bundles that shut genes down or start them
up without the help of proteins. Since the discovery of these RNA
clumps, called “riboswitches,” in 2002, scientists have been striving
to understand how they work and how they form. Now, researchers at
Stanford University are looking closer than ever at how the
three-dimensional twists and turns in a riboswitch come together by
grabbing it and tugging it straight. By physically pulling on this
loopy RNA, they have determined for the first time how a
three-dimensional molecular structure folds, step by step.
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.
Source: Stanford University
is also the extracellular matrix (ECM) which is made up of
glycosaminoglycans (protein sugar)and proteins such as collagen. This
ECM acts as a frame work for cells to interact and bind with. ECM is
particularly important in bone, cartilage and organs.