Part two of our nucleic
acid research coverage—see the Fall 2003 issue for an overview of DNA
research in the College of Chemistry.
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
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.
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.
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.
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.
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.)
Ribosome in Action
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
on image for larger view).
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).
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
“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,”
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).
on image for larger view).
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,”
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.
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.
RNA Shape Shifter
ribozyme structure from the Doudna lab (click on image for larger
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
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
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.
Doudna faculty page
Doudna Cate research website
Bustamante research site
Wemmer research site