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Surprisingly Versatile RNA

Part two of our nucleic acid research coverage—see the Fall 2003 issue for an overview of DNA research in the College of Chemistry.

In the world of the cell, it is the common wisdom that DNA carries information, while proteins perform chemical activities and make up the majority of cellular structures. RNA—ribonucleic acid—is typically viewed as the office grunt of the genetic world, transcribing the coded instructions of DNA for the assembly of amino acids into proteins. But RNA’s days of toiling in the shadows are coming to an end. It may not be on the cover of magazines and get the press coverage as its glamorous sister, DNA, but RNA is attracting more and more attention in scientific communities.

“I think that RNA is a far more interesting molecule than DNA,” said Ignacio Tinoco, Jr., who has worked with RNA for almost 40 years. “DNA is always double helical and acts as a storage facility, whereas RNA is much more versatile in the cell: it is involved in translation and in the transfer of amino acids. There is even a more recently discovered small interfering RNA that can change how the genome is read,” he explained.

RNA refuses to be pigeonholed. It shares with DNA the ability to store information—in fact some viruses don’t even have DNA at all, just RNA genomes. But RNA also shares with protein the ability to catalyze reactions.

Scientists in the College of Chemistry are furthering our knowledge of RNA and chemical biology. Using structural biology and biophysics, they hope to predict three dimensional structure and function from sequence, view the inner workings of complex molecular machinery and understand more about the fascinating properties of RNA.

A Bit of Background
First, if words like retrotransposon and hairpin loops seem like ordinary breakfast conversation, then you may skip ahead to the next section. For those of us more typical people who haven’t seen a biology textbook since high school (or maybe college), a bit of scientific background is in order.
The central dogma of molecular biology: DNA codes for RNA codes for protein. Okay, there are some notable exceptions to this paradigm (RNA codes for DNA in retroviruses, for one), but overall, it’s an accurate portrayal of how information is arranged hierarchically in the cell. DNA is the master copy of a cell’s instructions and is far too important to be allowed out of the safety of the nucleus.

To transmit its genetic information, DNA uses RNA as its intermediary. DNA is transcribed in the nucleus into messenger RNA (mRNA), which is a long string of ribonucleotide monomers. When the mRNA appears in the cytoplasm, the two subunits of the ribosome (a large protein and RNA complex) grab onto it and start moving down the string, translating the DNA’s directions and synthesizing protein.

The ribosome does this by reading the mRNA three nucleotides at a time and, as it is reading the code, recruiting the appropriate amino acids specified by the mRNA sequence. Once it has procured two amino acids, the ribosome holds them next to each other on its surface and RNA in the ribosome catalyzes the formation of a peptide bond. The ribosome continues to read the mRNA’s instructions, bringing in the correct amino acid, forming the peptide bond and increasing the length of the protein. These proteins then fold into their final structure and go on their way.

“The ribosome is a fascinating machine,” said Tinoco, a professor of chemistry in the graduate school. “The solving of the ribosome structure... is one of the most exciting developments in our field.” (Jamie Doudna Cate, a professor of chemistry and of molecular and cell biology, helped resolve the structure of the ribosome at the 5.5Å level as an assistant professor at the Whitehead Institute and MIT.)

The Ribosome in Action

Ribosomes are composed of two subunits: a large subunit (50S), shown on the right, and a small subunit (30S), shown on the left, in an image from Jamie Doudna Cate’s research group. The subunits are composed of long strands of RNA dotted with protein chains. When synthesizing a new protein, the two subunits lock together with a messenger RNA trapped in the space between. The ribosome then walks down the messenger RNA three nucleotides at a time, building a new protein piece-by-piece.

(H, head of the small subunit; B, small subunit body; CP, central protuberance of the large subunit. The arrow indicates the direction of movement in going from the open conformation to that in the intact ribosome).

(click on image for larger view).

“The ribosome is a ‘universal translator’,” observed Doudna Cate. “It can translate the four-letter code in DNA and its kissing cousin, messenger RNA, into the twenty-letter amino acid code of proteins.”

With such an important job to do, it is no surprise that the ribosome has not changed much in millions of years, with an extraordinarily uniform structure from bacteria to humans. But not an identical one. “Subtle differences between bacterial and human ribosomes allow us to use antibiotics that target bacterial ribosomes to treat infections,” said Doudna Cate. “However, bacteria are rapidly developing resistance to these antibiotics.”

To help develop new antibiotics, scientists are working to understand how the ribosome works and how antibiotics interfere with its activity. Doudna Cate and his group are working to record an atomic-resolution movie of the ribosome in action. “Ribosomes are dynamic; for example, their small and large ribosomal subunits associate and dissociate during one full cycle of protein synthesis,” he explained. Members of his lab have made frames of this movie at low resolution (9-10 Å) by using X-ray crystallography. They are now working to make the first frame of the movie at atomic resolution (3 Å, or about the length of two chemical bonds).

Stretching RNA
Carlos Bustamanate and Ignacio Tinoco also study RNA dynamics. Frequent collaborators (Tinoco served as Bustamante’s Ph.D. advisor), the two scientists love to stretch individual molecules, pulling them out straight, and watching them fold back again. “By mechanically manipulating individual molecules of RNA, we have learned a great deal about the thermodynamics and kinetics of the folding process,” said Bustamante. To stretch RNA (and other molecules), they attach each end of the target RNA to a polystyrene bead. They then use an “optical trap,” which consists of a laser beam holding and measuring the force on one bead as a piezoelectric actuator attached to the other bead supplies the nano-precise force necessary to unfold RNA.

“One of the advantages of single-molecule work is that we can follow the trajectory that a molecule adopts as it goes from folded to unfolded,” said Bustamante.

The kissing complex is an RNA tertiary structure formed by loop-loop interactions between two identical hairpins. The kissing hairpins respond to applied mechanical force like silly putty: when the force is increased slowly, the molecule is more elastic; but when the force is applied very fast, the entire structure becomes brittle. (from the Bustamante group).

(click on image for larger view).

“We were surprised to find that the same molecule, pulled multiple times, will follow a different pathway each time, although there are only a few prescribed paths for each molecule,” he continued. “Eventually, if we pull it enough times, we obtain a probability map of what pathways the molecule will follow.” This knowledge will help scientists to understand some of the critical aspects of folding—what architectural principles serve as the bedrock to RNA folding and therefore its function.

Beyond observing the folding process, Bustamante’s group is now synthesizing RNA with specific folding “intermediates” built in. “We have found eight barriers in refolding that oppose folding in a ribozyme—an RNA molecule that can catalyze a reaction. We can now study how the cell machinery behaves when encountering one of these predefined barriers by studying RNA helicase, the molecular motor that unwinds RNA.” His group has long suspected that the unfolding process is largely mechanical. “Now these synthetic RNA molecules can help us to confirm our suspicions.”
Bustamante and his laser tweezers have ushered in a new way of working with dynamics. “Previously scientists were stuck using bulk methods. Their efforts were frustrated by the large number of trajectories that were followed: they could only see the average. But we are working beyond that, with dynamics of individual molecules.

“Force is more biologically relevant than other methods of unfolding,” added Bustamante.

Tinoco uses the laser tweezers to dissect reactions one molecule at a time. “For example, we can study the ribosomal forces on the mRNA during protein translation or the force that DNA polymerase applies when opening DNA base pairs to synthesize a new DNA molecule.”

Tinoco wants to understand how sequence dictates structure and function. “One-dimensional beads-on-a-string RNA chains are folded into precise functional structures—from the sequence alone. We want to know exactly what folded, base-paired structure of an RNA is specified by the sequence. And how do RNA loops interact with each other, or with double-stranded regions to form compact three- dimensional structures.”

In addition to laser tweezers, Tinoco and his group use physical methods, such as multidimensional NMR, absorption and circular dichroism (spectroscopy based on the observation of left and right hand circular polarized light being absorbed slightly differently by matter) to determine the conformations and dynamics of nucleic acids.

Appearances are deceiving
RNA has long been thought of as nothing but an intermediary between DNA and the cell. Only recently has the wealth of activities that it’s involved in come to light. What enables RNA molecules to carry out their many biological tasks is the ability of their nucleotide strands or helices to fold themselves into complex three-dimensional structures. “We now know that RNA can carry out certain chemical reactions for which it also carries the code—bypassing both DNA and protein entirely,” said David Wemmer (Ph.D. ’79). What is not completely understood is how RNA performs this feat—a topic of intense study.

“We know that proteins carry out their functions through enzymes,” continued Wemmer, a professor of chemistry, “and we know that RNA also has enzymatic activity associated with it, but until recently no RNA enzymes had been looked at closely. The RNA enzyme we studied—one associated with cleavage of the RNA strand—was one of the first to be analyzed.”

Earlier, researchers had studied the area where cleavage takes place and had proposed a “hammerhead” model for the shape of the structure involved. Noted Wemmer, “Our studies confirmed that the hammerhead secondary structure was basically correct, and we have been able to add details about the folding that seems to precede cleavage.” Subsequent crystallographic work (for example from Bill Scott, Ph.D. ’92 now at UCSC) provided more information about the tertiary fold of this molecule.

The RNA Shape Shifter

HDV ribozyme structure from the Doudna lab (click on image for larger view).

And to end this article, let’s go back to the beginning—of life on Earth. How did life come about? “Chemical biologists have long suspected that RNA molecules were key to the process, in part because ribozymes—RNA molecules with enzyme-like activities—occur in many kinds of cells and viruses,” said Jennifer Doudna, a professor of chemistry and of molecular and cell biology.

Ribozymes differ from protein enzymes in that they both encode and chemically modify genetic information, implying that a primitive life form composed entirely of RNA might have preceded the evolution of modern protein- and DNA-dominated organisms.

In an ongoing effort to understand how ribozymes work as chemical catalysts and how they compare to their better-known protein enzyme counterparts, Doudna and her colleagues have been studying a small ribozyme harbored by the hepatitis delta virus. The hepatitis delta virus (HDV) ribozyme is required for processing long tandem copies of the viral RNA genome that form during rolling-circle replication of the virus in infected cells.

Ten new molecular structures of the HDV ribozyme, obtained using X-ray crystallography, show that the ribozyme twists its substrate—a single site within the viral RNA genome—into position and then uses an RNA nucleotide in the active site to break a chemical bond within the substrate. The ribozyme then changes its shape to release the cleaved substrate and prevent the chemical reaction from running in reverse. This shape-shifting ribozyme suggests that RNA structural rearrangements, in addition to other chemical strategies, may be widely used to control the reactivity of ribozymes in biology.

More to Discover
“I think we have a good understanding of many of the fundamentals of structural principles for nucleic acids,” said Wemmer. “However, there are undoubtedly many new ‘twists’ in how they work that remain to be discovered. It is not so many years ago that Berkeley alumnus Tom Cech discovered catalytic activity in RNA, which was a complete surprise (and thus worthy of a Nobel prize). New functions for RNA are still being discovered—for which the structural basis needs to be worked out.”

And surely, part of the excitement of research is that scientists don’t really know what is left out there to discover.

Related sites:

Jennifer Doudna faculty page

Jamie Doudna Cate research website

Carlos Bustamante research site

Ignacio Tinoco website

David Wemmer research site

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