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JAPAN NANONET BULLETIN
-- 92nd Issue -- March 22, 2007
Nanotechnology Researchers Network Center of Japan
Ministry of Education, Culture, Sports, Science and Technology (MEXT)
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IN THIS ISSUE
Nanonet Interview:
"RNA polymerase molecules sliding along DNA
-- Dynamics of biomolecules --"
Nobuo SHIMAMOTO, Professor, Structural Biology Center, National
Institute of Genetics, Research Organization of Information and Systems
Young Researchers' Introduction:
"Development and analysis of functional conducting organic materials"
Hideki YAMOCHI, Professor, Division of Molecular Materials Science
Research Center for Low Temperature and Materials Sciences, Kyoto
University
-- NANO CALENDAR --
For information on nanotechnology related symposiums and conferences
held in the world,
http://www.nanonet.go.jp/english/calendar/
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NANONET INTERVIEW
RNA polymerase molecules sliding along DNA
-- Dynamics of biomolecules --
(Issued in Japanese: June 7, 2006)
Nobuo SHIMAMOTO, Professor, Structural Biology Center, National
Institute of Genetics, Research Organization of Information and
Systems
"In molecular biology, biological phenomena used to be studied mainly
from functional aspects, but are now studied from mechanistic aspects
to solve the mechanisms by using the static structures of molecular
machines. I started researching nanobiology because of my interest in
the dynamic aspects of biological phenomena, especially those in which
physiological changes are governed by movements of one or two
biological molecules," Prof. Shimamoto says. He picked a transcription
system among such molecular machines and focused on the dynamic and
mechanical aspects of RNA polymerases on DNA. In other words, he tried
to clarify how transcription, which is the transfer of genetic
information from DNA into RNA, is described in terms of the motions of
molecules and molecular domains using newly emerging technology, such
as single molecule measurements.
RNA polymerase works in the form of a single molecule that transcribes
a gene existing as one or several copies, and each molecular machine
provides a different blueprint for protein synthesis according to the
gene. It has been known that all RNA polymerases synthesize short RNAs
in addition to the full-length RNAs that act as a blueprint.
Researchers had thought that these shorter RNAs were failed or aborted
precursors of the full-length RNA, assuming a sequential pathway of
transcription initiation composed of the chemical steps that were
believed to be essential at that time. However, when Shimamoto's team
compared the time courses of the synthesis of short and full-length
RNAs by using E. coli RNA polymerase, which is the only fully
reproduced artificial system, composed of purified proteins at present,
they found that the shorter RNAs are continuously synthesized even
after completion of the full-length RNA synthesis. This means that
there is a distinct transcription machine that only produces short
RNAs and that the initiation of RNA synthesis is branched into two or
more pathways according to different molecular machines. One branch
leads to normal synthesis of the full-length RNA, and the other leads
to an arrest accompanied with the synthesis of the shorter RNAs. The
team named the newly found machine "moribund complex," and the
different machines are produced from homogeneous preparations of RNA
polymerase and DNA. They experimentally proved that there is indeed a
branched initiation mechanism and showed that different machines have
different conformations of proteins and DNA (Fig.1).
Prof. Shimamoto says, "The moribund complex may be thought to be
useless, but in fact, it regulates transcription. Proteins, called
GreA and GreB, which have been found as transcriptional activators
working in elongation of RNA, activate initiation by converting the
moribund complex into a productive complex." Historically, we tend to
assume that a biological reaction is a sequence of chemically
essential processes, but this study shows that a seemingly useless
process, i.e., abortive synthesis in this case, can have an essential
role for life. He says, "Regulation is the core heart of biology, and
living organisms even utilize a process that we superficially
considered useless according to our limited knowledge. This study was
a good lesson for me."
Prof. Shimamoto also addressed the mechanism in which RNA polymerases
identify the DNA segment to initiate transcription, namely, a promoter.
How does RNA polymerase find a promoter that is merely a small part of
huge chromosomal DNA? He observed how the molecules of DNA-binding
proteins move on DNA by using single molecule imaging and proved that
E. coli RNA polymerase can slide along DNA. He fixed extended T7 phage
DNA in parallel on a slide glass, where both ends of the DNA are fixed,
but the middle is free for the protein to access, and then, a solution
containing fluorescently-labeled RNA polymerase molecules is added to
the slide at an angle to the fixed DNA (Fig.2).
Prof. Shimamoto says, "When the single RNA polymerase molecules that
drift with the bulk flow reach the DNA fixed in parallel, we observed
that their movements were parallel to the DNA, and such movements
would not be possible if the protein molecule were not sliding." This
is the first direct evidence of a protein sliding along DNA by single-
molecule dynamics. His team also experimentally proved that RNA
polymerase can track a helical groove of DNA during sliding by
rotating around the DNA double helix, which is the proof that RNA
polymerase can read a DNA sequence as a human does, by introducing a
nano-tachometer (Fig.3). He says, "This is an excellent mechanism by
which RNA polymerase selects the double-stranded DNA from huge excess
of RNA that does not have the groove in a cell."
The longer a DNA molecule, the more frequently a protein meets the DNA
to start sliding, if the protein can slide along DNA. Such a mechanism
allows the protein to reach its target sequence faster. Prof.
Shimamoto focused on E. coli tryptophan repressor (TrpR) and P. putida
CamR, which are typical bacterial repressors that inhibit
transcription by binding to their target sequences. Since most
sequences on DNA weakly catch these DNA binding proteins, such weak
binding sites should compete for catching proteins against the target
sequences. This means that there must be a critical number of protein
molecules in a cell to bind to the target sequence. According to
previous reports, the number of TrpR molecules is 100-fold less than
the critical number, and thus, how can it inhibit transcription? He
experimentally showed that these repressors slide along DNA in their
association process to their targeted sequences, while they dissociate
from their targets without sliding after binding to the targets.
Consequently, as the DNA becomes longer, the stability of TrpR-target
complex becomes higher. He named the effect of DNA length on the
stability of a DNA-protein complex "antenna effect" because an antenna
either catches a weak signal from air or senses objects in insects
(Fig.4). Sliding is one of the mechanisms that exert an antenna
effect; although this mechanism is sometimes misunderstood as a
violation of a thermodynamic rule called "detailed balancing," careful
consideration of time ranges of the involved physical processes showed
that this antenna effect does not contradict thermodynamics.
Prof. Shimamoto says, "Nanobiology is a discipline to find out how
motions of molecules in cells generate macroscopic phenomena, while
nanobiotechnology is a discipline to create molecular machinery by
utilizing biomolecules. So, collaboration between nanobiology and
nanobiotechnology is required." However, he is strongly against so-
called commercialization-oriented basic research. "In principle, basic
research can be promoted only by small but widely distributed funds,
and commercialization cannot be promoted by such funds. For
commercialization, product-oriented projects must be essential, a
large sum of money must be invested in the projects, and then, they
must be strategically carried out in consideration of cost-performance.
This is why basic research and commercialization cannot be directly
combined. They require different strategies. We must train specialists
for combining these two different components, which is more trans-
disciplinary than the field called translational research (Fig.5)." He
adds, "To receive research grants, some may push their research as a
work that can be commercialized, but neither basic scientists nor
people involved in commercialization are able to notice, if the
research is original. Only specially trained people are able to notice
such possibilities. If nanobiology is used for commercialization-
oriented basic research, it will go nowhere. So, the correct way of
funding and the need for specialists are essential, and I have to keep
saying this opinion out loud."
(Interviewer: Kuniko Ishiguro, Cosmopia Inc.)
For more information including figures,
http://www.nanonet.go.jp/english/mailmag/2007/092a.html
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YOUNG RESEARCHERS' INTRODUCTION
Development and analysis of functional conducting organic materials
(Issued in Japanese: August 30, 2006)
Hideki YAMOCHI, Professor, Division of Molecular Materials Science
Research Center for Low Temperature and Materials Sciences, Kyoto
University
Since 2002, our research center has performed two missions: the stable
supply of cryogens in Kyoto University and our own original scientific
research. The facilities for the former purpose are being set up,
while those for the research are not yet organized. Although all of
the scientific works have been carried out with the help of other
departments, the investigations in our center are highly original.
Our division investigates functional materials based on low molecular
weight organic molecules. Focusing mainly on the conducting properties,
structural and physical properties have been investigated to develop
new materials and new concepts. For example, the study on
ethylenedioxytetrathiafulvalene (EDO-TTF) is presented below.
An investigation aimed at the partial suppression of the self-
assembling nature of bis(ethylenedioxy)tetrathiafulvalene (BEDO-TTF),
from which complexes with stable metallic states have been afforded
almost exclusively. The analogue EDO-TTF did not show the ability to
self-assemble. Instead, a peculiar metal-insulator transition
associated with a distinct molecular deformation was observed for (EDO
-TTF)2PF6 at around 280 K. The mechanism was interpreted as
cooperation among Peierls, charge ordering, and anion ordering
transitions (Fig. 1). Such a concept of mechanism mixing has been
rarely considered. From the viewpoint of multi-instability, the photo
-induced phase transition (PIPT) was examined in cooperation with
other research groups. A pulsed weak laser light was used to
stimulate the insulating phase, and ca. 500 donor molecules were
converted into the highly conducting metastable state within 1.5 ps
(Fig. 2). The ultra-fast and highly efficient conversion can be used
as the basis of optoelectronics materials in the future as well as the
seeds for basic sciences. It is noteworthy that other research groups
have also examined the PIPT for conducting charge-transfer complexes
after our reports were published. At present, cooperative work is
underway to elucidate the dynamics of the PIPT. Also, new materials
are being explored under the working hypothesis that flexible and
strongly correlated pi-electron systems will provide conductors having
multi-instabilities.
For more information including figures,
http://www.nanonet.go.jp/english/mailmag/2007/092b.html
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