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August 20, 2004

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Interactions between viral and bacterial proteins promise new directions for antibiotics University.
Rockefeller University
How does the molecular machine responsible for activating To interview Rockefeller University scientists or to request
scientists, led by Milton
genes choose which gene to switch on, from among the photos, contact media relations manager Katherine Fenz
H. Werner, Ph.D., were
at 212-327-7913 or by email.
30,000 genes contained in each cell of the human body? able to visualize the
interaction between
In the August 4 issue of the EMBO Journal, researchers at
two proteins, AsiA (red)
Rockefeller University report that they are beginning to answer and sigma70 (blue),
that question in bacteria, and the answers are not only which causes the loss
surprising, but may also aid in the development of powerful of gene expression in
new antibiotics. the bacterium E. coli.
Drugs exploiting AsiA’s
If the current research is extended, “we could have a new ability to inhibit gene
strategy for developing an antibacterial agent, one that would activation could hold
act to weaken a protein’s structure,” says Milton H. Werner, promise as antibiotics
Ph.D., the Rockefeller scientist leading the study. and anti-cancer
therapies
“The real value in these ndings is that we are learning new
ideas about how you would make an antibiotic that would kill off bacteria in the same way that
nature’s killers of bacteria already do,” says Werner, associate professor and head of the Laboratory
of Molecular Biophysics at Rockefeller. “If we could deliver just those proteins that inhibit bacterial
transcription, we would have a truly powerful antibiotic. I am very excited about just that idea.”

The study visualizes an interaction between phage, a virus that infects bacteria, and the proteins
that make up the bacterium. The molecular image shows that the structure of the bacterium’s
protein is altered as a consequence of the interaction with the phage virus, and the result is both an
inhibition of gene activation in bacteria and an increase in the expression of the phage’s genes.

“It is very unexpected that proteins controlling gene expression can do so by signi cantly altering
the organization of an enzyme,” says Werner. “In this case it leads to the loss of all gene expression
in E. coli.”

In the bacterium E. coli, proteins called sigma factors monitor the environment of the microbe’s cell
and promote the activation, or expression, of certain genes by recruiting a molecular machine
called RNA polymerase. Each sigma factor is like a key, and is only able to activate the speci c
subset of genes that it ts. The sigma factors bring the RNA polymerase to the gene promoters, the
segment of DNA that is the start site to transcribe the gene’s instructions. The seven sigma factors
present in E. coli can be divided into two families. One of these families, called Sigma70, unlock
“housekeeping” genes, which are essential for metabolism and survival of bacteria.

When the phage enters an E. coli cell, it rst produces a set of proteins that inhibits all bacterial
transcription. With the help of sigma70, the phage hijacks the bacterial RNA polymerase and forces
it to only switch on, or transcribe, phage genes.

In a 2001 EMBO Journal paper, Werner and colleagues described the structure of AsiA and
suggested that AsiA, expressed as a dimer (two subunits joined together) dissociates to interact
with RNA polymerase. The current paper illustrates how AsiA interacts with sigma70 and remodels
the domain of sigma70 with which it interacts.

“AsiA in uences the polymerase in such a way that it cannot recognize the bacterial promoters
anymore,” says Werner, “but instead recognizes a whole new set of phage promoters as a
consequence. By understanding the interaction between AsiA and sigma70, which leads to the loss
of bacterial gene expression, we can mimic it.”

Antibiotics that interfere with a gene’s transcription of its instructions to a body cell are of great
interest because they are less likely to become ineffective. Normally, simple mutations can render a
bacterium resistant to an antibiotic. However, transcription depends on the ability of proteins like
sigma70 to recognize the promoter DNA, so any mutations in sigma70 must be followed by
changes in the DNA. The chances that compatible mutations occur in both the lock and the key to
confer antibiotic resistance are very slim, Werner says.

Understanding the function of a protein like AsiA in transcription aids in the development of
species-speci c antibiotics against diseases such as tuberculosis. In addition, while AsiA is the
product of a bacterial pathogen, the same kind of strategy could also be employed by pathogens of
animals and humans. Drugs expanding on AsiA’s ability to inhibit transcription could hold promise
as anti-cancer therapies as well.

Research by Seth Darst, Ph.D., another Rockefeller University researcher, has examined the
interaction between sigma70 and various inhibitory proteins called anti-sigma factors. Anti-sigma
factors serve to prevent sigma proteins from interacting with RNA polymerase until the proper time,
effectively blocking the sigma key from tting into its lock.

In this new research paper, Werner and Rockefeller colleagues Lester J. Lambert and Yufeng Wei
were able to visualize the interaction between AsiA and sigma70 using a spectroscopic technique
called nuclear magnetic resonance (NMR) spectroscopy. They show that in contrast to other anti-
sigma factors, when AsiA binds to sigma70, it remodels the key completely, so that now sigma70
only ts phage genes. Sigma70’s new orientation most likely causes subsequent changes in the
shape of the RNA polymerase, though that question is still under investigation.

AsiA is now only one of three activators whose interaction with the polymerase has been visualized.
Transcription factors are notoriously hard to crystallize because they have so many disordered
areas. Werner’s and others’ use of NMR spectroscopy is nally bringing transcription factor
structures into light.

“We have almost no pictures of how transcription factors engage polymerase. Lots and lots of
people have tried, but they are not amenable to crystallization because they tend to have disordered
segments,” explains Werner. “We have an advantage that we can make measurements from
proteins that are poorly structured, disordered or unstable, and very often get very useful structural
information from. Disorder doesn’t disturb us.”

Co-authors include Virgil Schirf, Ph.D., and Borries Demler, Ph.D., at the University of Texas Health
Science Center.

This research was supported by grants from the National Institutes of Health and National Science
Foundation. Werner is a W.M. Keck Foundation Distinguished Young Scholar.

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Researcher studying the dynamics of gene

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