New key to organism complexity identified
(Photo by Roy Kaltschmidt, Berkeley Lab) |
The
enormously diverse complexity seen amongst individual species within the animal
kingdom evolved from a surprisingly small gene pool. For example, mice
effectively serve as medical research models because humans and mice share
80-percent of the same protein-coding genes. The key to morphological and
behavioral complexity, a growing body of scientific evidence suggests, is the
regulation of gene expression by a family of DNA-binding proteins called
"transcription factors." Now, a team of researchers with the U.S.
Department of Energy's Lawrence Berkeley National Laboratory (Berkeley Lab) and
the University of California (UC) Berkeley has discovered the secret behind how
one these critical transcription factors is able to perform -- a split
personality. Using a technique called single-particle cryo-electron microscopy,
the team, which was led by biophysicist Eva Nogales, showed that the
transcription factor known as "TFIID" can co-exist in two distinct
structural states. These two states -- the canonical and the rearranged --
differ only in the translocation of a single substructural element -- known as
lobe A -- by 100 angstroms (an atom of hydrogen is about one angstrom in
diameter). This structural shift enables initiation of the transcription
process by which the genetic message of DNA is copied to RNA for the eventual
production of proteins.
"TFIID
by itself fluctuates between the canonical and rearranged states," Nogales
says. "When TFIID becomes bound to another transcription factor, TFIIA, it
shifts mostly to the canonical state, but in the presence of both TFIIA and
DNA, the TFIID shifts to the rearranged state, which enables recognition and
binding to key DNA sequences and marks the start of the transcriptional
process."
Understanding
the reorganization of TFIID and its role in transcription provides new insight
into the regulation of gene expression, Nogales says, a process critical to the
growth, development, health and survival of all organisms.
Nogales is a leading authority on electron microscopy and holds
joint appointments with Berkeley Lab, the University of California (UC) at
Berkeley, and the Howard Hughes Medical Institute (HHMI). She is the
corresponding author of a paper describing this research in the journalCell, titled
"Human TFIID Binds to Core Promoter DNA in a Reorganized Structural
State." Co-authors are Michael Cianfrocco, George Kassavetis, Patricia
Grob, Jie Fang, Tamar Juven-Gershon and James Kadonaga.
The growing number of organisms whose genomes have been
sequenced and made available for comparative analyses shows that the total
number of genes in an organism's genome is no measure of its complexity. The
fruit fly, Drosophila, for example, is far
more complex than the nematode worm, Caenorhabditis elegans, but has
about 6,000 fewer genes than the worm's 20,000. The total number of human genes
is estimated to fall somewhere between 30,000 and 40,000. By comparison, the
expression of the genes of both the fruit fly and the nematode are regulated
through about 1,000 transcription factors, whereas the human genome boasts
approximately 3,000 transcription factors. That multiple transcription factors
often act in various combinations with one another creates even more
evolutionary roads to organism complexity.
"Although
the number of protein coding genes has remained fairly constant throughout
metazoan evolution, the number of regulatory DNA elements has increased
dramatically," Nogales says. "Our discovery of the existence of two
structurally and functionally distinct forms of TFIID suggests a potential
molecular mechanism by which a combination of transcription factors can tune
the expression level of genes and thereby give rise to a diversity of
outcomes."
Despite
its critical role in transcription, high-resolution structural information of
TFIID has been restricted to crystal structures of a handful of protein
subunits and domains. Nogales and her colleagues are the first group to obtain
three-dimensional visualization of human TFIID that is bound to DNA. The
single-particle cryo-electron microscopy technique they employed records a
series of two-dimensional images of an individual molecules or macromolecular
complexes frozen in random orientations, then computationally combines these
images into high-resolution 3D reconstructions.
"Through cryo-EM and extensive image-sorting, we found that
TFIID exhibits a surprising degree of flexibility, moving its lobe A, a region
that covers approximately one-third of the complex, by 100 angstroms across its
central channel," says Cianfrocco, lead author of the Cell paper.
"This movement of the lobe A is absolutely essential for TFIID to bind to
DNA."
Nogales
says that while many macromolecular complexes are known to be flexible, this
typically involves the limited movement of a small region within the complex,
or some tiny motion of the entire complex. The movement of TFIID's lobe A
represents an entire restructuring that dramatically alters what the molecule
can do. In the canonical state, TFIID's lobe A is bound to its lobe C, which
appears to be the preferred form of free TFIID. In the rearranged state,
TFIID's lobe A is bound to its lobe B, which is the state in which it can then
strongly bind to DNA promoters.
"The
TFIIA molecule serves as the mediator for this transition, maintaining TFIID in
the canonical state in the absence of DNA and initiating the formation of the
rearranged state in the presence of promoter DNA," Cianfrocco says.
"Without the presence of TFIIA, the binding of TFIID to DNA is very
weak."
Nogales
and her colleagues are now studying how TFIID, once it is bound to DNA,
recruits the rest of the machinery required to transcribe the genetic message
into RNA.
"Our
new work will involve constructing a macromolecular complex that is well over
two million Daltons in size, which is about the size of a bacterial
ribosome," Nogales says. "The size and relative instability of our complex
will represent a major experimental challenge."
This
work was supported by grants from the National Institutes of Health and the
Human Frontier in Science Program in Strasbourg, Germany, and the Howard Hughes
Medical Institute.
Source: DOE/Lawrence
Berkeley National Laboratory
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