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Topic Name: Stanford researchers have determined for the first time how a three-dimensional molecular structure folds, step by step

Category: Genetic Engineering

Research persons: Research Team

Location: Stanford University, United States

Details

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.

Note for Riboswitch
In molecular biology, a riboswitch is a part of an mRNA molecule that can directly bind a small target molecule, and whose binding of the target affects the gene's activity. Thus, an mRNA that contains a riboswitch is directly involved in regulating its own activity, depending on the presence or absence of its target molecule.
Although the metabolic pathways in which some riboswitches are involved have been studied for decades, the existence of riboswitches has only been relatively recently discovered, with the first experimental validations of riboswitches being published in 2002. This oversight may relate to an earlier assumption that genes are regulated by proteins, not by the mRNA transcript itself. Now that riboswitches are a known mechanism of genetic control, it is reasonable to speculate that more riboswitches will be found.
Riboswitches are conceptually divided into two parts: an aptamer and an expression platform. The aptamer directly binds the small molecule, and the expression platform undergoes structural changes in response to the changes in the aptamer. The expression platform is what regulates gene expression.
Expression platforms typically turn off gene expression in response to the small molecule, but some turn it on. Expression platforms include:
The formation of rho-independent transcription termination hairpins 
Folding in such a way as to sequester the ribosome-binding site, thereby blocking translation 
Self-cleavage (i.e. the riboswitch contains a ribozyme that cleaves itself in the presence of sufficient concentrations of its metabolite) 
Folding in such a way as to affect the splicing of the pre-mRNA. A TPP riboswitch in Neurospora crassa (a fungus) controls alternative splicing to conditional produce a uORF, thereby affecting expressing of downstream genes.

Note for Crystallography
Crystallography is the experimental science of determining the arrangement of atoms in solids. In older usage, it is the scientific study of crystals.
Before the development of X-ray diffraction crystallography (see below), the study of crystals was based on the geometry of the crystals. This involves measuring the angles of crystal faces relative to theoretical reference axes (crystallographic axes), and establishing the symmetry of the crystal in question. The former is carried out using a goniometer. The position in 3D space of each crystal face is plotted on a stereographic net, e.g. Wulff net or Lambert net. In fact, the pole to each face is plotted on the net. Each point is labelled with its Miller index. The final plot allows the symmetry of the crystal to be established.
Crystallographic methods now depend on the analysis of the diffraction patterns that emerge from a sample that is targeted by a beam of some type. The beam is not always electromagnetic radiation, even though X-rays are the most common choice. For some purposes electrons or neutrons are used, which is possible due to the wave properties of the particles. Crystallographers often explicitly state the type of illumination used when referring to a method, as with the terms X-ray diffraction, neutron diffraction and electron diffraction.
These three types of radiation interact with the specimen in different ways. X-rays interact with the spatial distribution of the valence electrons, while electrons are charged particles and therefore feel the total charge distribution of both the atomic nuclei and the surrounding electrons. Neutrons are scattered by the atomic nuclei through the strong nuclear forces, but in addition, the magnetic moment of neutrons is non-zero. They are therefore also scattered by magnetic fields. Because of these different forms of interaction, the three types of radiation are suitable for different crystallographic studies.

Note for RNA Polymerase
RNA polymerase (RNAP or RNApol) is an enzyme that makes an RNA copy of a DNA or RNA template. In cells, RNAP is needed for constructing RNA chains from DNA genes, a process called transcription. RNA polymerase enzymes are essential to life and are found in all organisms and many viruses. In chemical terms, RNAP is a nucleotidyl transferase that polymerizes ribonucleotides at the 3' end of an RNA transcript.
RNAP was discovered independently by Sam Weiss and Jerard Hurwitz in 1960. By this time the 1959 Nobel Prize in Medicine had been awarded to Severo Ochoa and Arthur Kornberg for the discovery of what was believed to be RNAP, but instead turned out to be polynucleotide phosphorylase.
The 2006 Nobel Prize in Chemistry was awarded to Roger Kornberg for creating detailed molecular images of RNA polymerase during various stages of the transcription process.
RNAP can initiate transcription at specific DNA sequences known as promoters. It then produces an RNA chain which is complementary to the template DNA strand. The process of adding nucleotides to the RNA strand is known as elongation; In eukaryotes, RNAP can build chains as long as 2.4 million nucleosides (the full length of the dystrophin gene). RNAP will preferentially release its RNA transcript at specific DNA sequences encoded at the end of genes known as terminators.
Products of RNAP include:
Messenger RNA (mRNA)—template for the synthesis of proteins by ribosomes. 
Non-coding RNA or "RNA genes"—a broad class of genes that encode RNA that is not translated into protein. The most prominent examples of RNA genes are transfer RNA (tRNA) and ribosomal RNA (rRNA), both of which are involved in the process of translation. However, since the late 1990s, many new RNA genes have been found, and thus RNA genes may play a much more significant role than previously thought. 
Transfer RNA (tRNA)—transfers specific amino acids to growing polypeptide chains at the ribosomal site of protein synthesis during translation 
Ribosomal RNA (rRNA)—a component of ribosomes 
Micro RNA—regulates gene activity 
Catalytic RNA (Ribozyme)—enzymatically active RNA molecules 
RNAP accomplishes de novo synthesis. It is able to do this because specific interactions with the initiating nucleotide hold RNAP rigidly in place, facilitating chemical attack on the incoming nucleotide. Such specific interactions explain why RNAP prefers to start transcripts with ATP (followed by GTP, UTP, and then CTP). In contrast to DNA polymerase, RNAP includes helicase activity, therefore no separate enzyme is needed to unwind DNA.

Other co-authors of the paper, "Direct Observation of Hierarchical Folding in Single Riboswitch Aptamers," are Stanford graduate students William J. Greenleaf and Kirsten Frieda; Daniel A. N. Foster of the University of Alberta; and Michael T. Woodside of the University of Alberta and the National Institute for Nanotechnology, National Research Council of Canada.

Funding was provided by the National Institute of General Medical Sciences and the National Institute for Nanotechnology at the University of Alberta.