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RNA Mediation of Protein Synthesis,
Protein Export and Gene Regulation
RNA molecules are uniquely capable of encoding and controlling the
expression of genetic information, often as a consequence of their
three-dimensional structures. We are interested in understanding
RNA-mediated initiation of protein synthesis, and RNA-protein
complexes involved in targeting proteins for export out of cells.
We are also investigating the early steps in gene regulation by
RNA interference.
Internal Ribosome Entry Site (IRES) RNAs
Most eukaryotic and viral messages initiate translation by a
mechanism involving recognition of a 7-methylguanosine cap at the 5'
end of the mRNA. In a few cases, however, translation occurs via a
cap-independent mechanism in which an Internal Ribosome Entry Site
(IRES) in the 5' untranslated region of the mRNA recruits the
ribosome. In Hepatitis C virus (HCV), the ~400 nucleotide IRES
folds into a magnesium-dependent structure
in which loops thought to interact with the ribosome are exposed on
the surface of the RNA. Point mutations that destroy IRES activity
disrupt the folded structure of the RNA. The IRES is formed from two
independently folding structural domains. One of these, the "core",
binds specifically to the 40S subunit of eukaryotic ribosomes, while
the other domain interacts with initiation factor eIF3. Structures
of the IRES-40S subunit complex, determined by cryo-electron microscopy
in collaboration with Joachim Frank
(HHMI, Health Research Inc., Wadsworth Center), revealed that the
IRES induces a significant conformational change in the 40S subunit
upon binding. This conformational change helps lock the start of
the viral mRNA protein coding sequence into the correct site on the
40S subunit.
In collaboration with Eva Nogales
(HHMI, UC Berkeley), we determined structures
of the HCV IRES in complex with the human translational machinery,
showing how the IRES can functionally replace proteins that help
position most cellular mRNAs on the ribosome. Using affinity
purified samples, mass spectrometry has revealed the full composition
and post-translational modification states of IRES-bound complexes that
assemble in human cell extracts (in collaboration with
Julie Leary,
UC Davis). Working with
Carol Robinson
at Cambridge University, we are analyzing intact IRES-ribosome
complexes by mass spectrometry to determine how the HCV IRES induces
assembly of active human 80S ribosomes. (This work is supported in
part by the NIH.)
In related experiments, we have discovered a class of IRES RNA
sequences in yeast that activate translation of genes in response
to starvation, enabling protein expression under conditions where
most mRNAs are translationally silenced. Unlike viral IRESs, the
yeast IRESs do not require three-dimensional RNA structures to
recruit the ribosome, although recent data hint that RNA structure
might regulate IRES activity. Exciting experimental data show that
yeast IRES function is essential for invasive growth, the developmental
pathway that haploid yeast enter in response to starvation. Current
work focuses on elucidating the mechanism of these cellular IRES RNAs,
which appears to be distinct from that of viral IRES elements, and
determining whether this kind of RNA-mediated translational control
is conserved in higher eukaryotes.
View an annimation of the IRES RNA at work (6.1MB)
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Structure and Mechanism of the Signal Recognition Particle (SRP)
The signal recognition particle (SRP) is a highly conserved
ribonucleoprotein responsible for transport of nascent polypeptides
targeted for secretion or membrane insertion. In prokaryotes, the SRP
consists of one protein (Ffh) and one RNA molecule (4.5S RNA), and both
are required for SRP activity. The RNA sequence corresponding to the
Ffh binding site has been maintained through evolution, and is virtually
identical in organisms from the three kingdoms of life - bacteria,
archaea and eukaryotes. The RNA plays a key, yet undetermined, role
in the protein targeting pathway. In 2000 we determined the
crystal structure of the complex
at 1.5 Å resolution, revealing a fascinating network of contacts
at the RNA-protein interface that explain the observed evolutionary
conservation. Using site-directed hydroxyl radical probing, we
discovered that the association of the SRP with its receptor triggers
a dramatic conformational change in the complex, localizing the SRP RNA
and the adjacent signal peptide binding site at the SRP-receptor
heterodimer interface. The orientation of the RNA explains how peptide
binding and GTP hydrolysis can be coupled through direct structural
contact during cycles of SRP-directed protein translocation. Recent
experiments show that the position of the SRP RNA within the SRP-receptor
complex enhances the rate of GTP hydrolysis in the complex above a
critical threshold required in vivo. Work towards a crystal
structure of the SRP-receptor complex (a ~130 kD assembly) has been
aided by the selection and purification of multiple antibody proteins
using phage display technology.
View an annimation of the SRP at work (8.7MB)
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RNA Recognition by Dicer Enzymes
Double-stranded RNA induces potent and specific gene silencing
in a broad range of eukaryotic organisms. This mode of gene
silencing, called RNA interference (RNAi), acts at the
transcriptional level through formation of heterochromatin and
at the post-transcriptional level through mRNA degradation and
translational suppression. In all cases, RNAi begins with the
processing of endogenous or introduced precursor RNA into micro-RNAs
(miRNAs) and small interfering RNAs (siRNAs) 21-25 nucleotides
in length by the enzyme Dicer. We recently solved the
crystal structure
of an intact Dicer enzyme, revealing how Dicer functions as a
molecular ruler to measure and cleave duplex RNAs of a specific
length. The structure has now been refined to higher resolution,
and a series of mutant forms of Dicer have been used to delineate
the roles of various domains and interactions both in vitro
and in vivo. Ongoing work focuses on determining how Dicer
interacts with other components of the RNAi pathway and how diced
RNAs are targeted to specific mRNAs. (This work is supported by the NIH.)
Read about dicer in
Chemical and Engineering News 2006 Highlights
(2.0MB .pdf)
View an annimation of Dicer at work (7.7MB)
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