The turn of the corkscrew : Structural analysis uncovers mechanisms of gene expression

The turn of the corkscrew : Structural analysis uncovers mechanisms of gene expression

  9:36:00 PM    Science Lover

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The diverse functions of living cells are all based on the information encoded in the structure of the hereditary material DNA. Gene expression must therefore be tightly controlled, and this task is accomplished by the binding of regulatory proteins to, and their removal from, specific DNA sequences. One class of large molecular machines known as Swi2/Snf2 remodelers plays a central role in modulating these processes. However, until now, it was not clear how Swi2/Snf2 remodelers actually work. A team led by Professor Karl-Peter Hopfner at the Gene Center at Ludwig-Maximilians-Universität (LMU) in Munich has clarified the structure and function of the remodeler Mot1 (Modifier of Transcription 1), which binds directly to DNA. It turns out that Mot1 acts like a molecular corkscrew that migrates along the DNA, following its helical contour. During its progress, Mot1 displaces a crucial transcription factor called TBP (for “TATA Box Binding Protein”) from the DNA. Removal of TBP from a TATA box represses transcription of the adjacent gene, and the protein encoded by that gene is no longer synthesized. At the same time, TBP is stabilized and its binding specificity is changed, which facilitates the expression of genes that lack TATA boxes and code for other proteins. (Nature 6 July 2011)

The DNA in the cells of higher organisms is tightly wrapped around protein complexes called nucleosomes. This type of structural organization not only makes it possible to package the long DNA molecules in a highly compact form, it also provides the basis for the controlled expression of genetic information. Densely packed sections of the molecule are effectively in a repressed state, and genes located in these DNA segments cannot be transcribed. Activation of repressed genes depends on the intervention of complex molecular machines, so-called Swi2/Snf2 remodelers, which reorganize condensed stretches of DNA so as to make them accessible for transcription. The precise mode of action of remodelers has so far been unclear, mainly because most of them are made up of several components and the active complexes are difficult to study. This is why Hopfner chose to study Mot1, which is a comparatively simple representative of the family that functions as an Swi2/Snf2 remodeler on its own, and can serve as a guide to understanding the more complicated members of the class. Mot1 is known to participate in the control of gene expression, but how exactly it does so is not well understood.

The first stage of the process that leads to the synthesis of a given protein is the transcription of the specific segment of DNA that codes for it into molecules of messenger RNA. This initial step requires the action of so-called transcription factors. One of the most important of these is TBP, which binds preferentially to DNA sequences called TATA boxes that are located near the beginnings of many genes. Binding of TBP introduces a kink into the DNA, and this landmark serves as a platform for the binding of further proteins, ultimately leading to the assembly of the complex necessary for the initiation of transcription. Mot1 regulates transcription by actively removing TBP from the DNA, using ATP as a source of energy. “How Mot1 dissociates the TBP-DNA complex was completely unclear up to now,” says Hopfner. With the aid of so-called hybrid methods - in which data obtained from high-resolution X-ray diffraction analysis of the crystallized protein complex with images of the same molecular complex taken with the electron microscope were combined - Hopfner’s team was able to define the three-dimensional structure of the Mot1-TBP complex for the first time. This revealed how Mot1 recognizes the surface of the DNA-bound TBP. “Once Mot1 has recognized TBP, it binds to the adjacent DNA and begins to migrate along the DNA strand, using the energy released by the hydrolysis of ATP to power its movement. This helical movement, which is reminiscent of the insertion of a corkscrew, causes TBP to detach from the DNA,” explains Dr Petra Wollmann, who is first author on the new study. The researchers were surprised to find that Mot1 contains a strikingly extended loop. After TBP has dissociated from the DNA, this loop masks TBP’s DNA binding site and prevents the protein from reoccupying it.



Previous studies had reported what appeared to be paradoxical observations, which indicated that Mot1 inhibits transcription of TATA box-containing genes while facilitating the expression of genes that lack canonical TATA boxes. “Our results suggest that Mot1 also stabilizes the DNA-free conformation of TBP, increasing the probability that it can reach, bind to and activate genes that lack TATA boxes,” explains Hopfner. In other words, Mot1 is also a redistribution factor, which enables TBP to bind to different sequences and thus controls its association with other cellular components. This combination of detachment and redistribution functions may be a common feature of remodeling complexes, and would help to explain how they mediate the large-scale redistribution of DNA-binding regulatory proteins. (göd/PH)

The project was carried out under the auspices of two Clusters of Excellence - the Center for Integrated Protein Science Munich (CiPSM) and the Munich Centre for Advanced Photonics (MAP). The work was also supported by the German Research Foundation (DFG) as part of the Collaborate Research Centers (SFB) 646 and TR5, and by the LMUexcellent Investment Fund.

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Researchers decipher protein structure of key molecule in DNA transcription system

Researchers decipher protein structure of key molecule in DNA transcription system

  11:59:00 PM    Science Lover

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The research adds an important link to discoveries that have enabled scientists to gain a deeper understanding of how cells translate genetic information into the proteins and processes of life. The findings, published in the July 3 advance online issue of the journal Nature, were reported by a research team led by Yuichiro Takagi, Ph.D., assistant professor of biochemistry and molecular biology at Indiana University School of Medicine.

Scientists have deciphered the structure of an essential 
part of Mediator, a complex molecular machine that plays 
a vital role in regulating the transcription of DNA.

The fundamental operations of all cells are controlled by the genetic information – the genes –stored in each cell's DNA, a long double-stranded chain. Information copied from sections of the DNA – through a process called transcription – leads to synthesis of messenger RNA, eventually enabling the production of proteins necessary for cellular function. Transcription is undertaken by the enzyme called RNA polymerase II.

As cellular operations proceed, signals are sent to the DNA asking that some genes be activated and others be shut down. The Mediator transcription regulator accepts and interprets those instructions, telling RNA polymerase II where and when to begin the transcription process.

Mediator is a gigantic molecular machine composed of 25 proteins organized into three modules known as the head, the middle, and the tail. Using X-ray crystallography, the Takagi team was able to describe in detail the structure of the Mediator Head module, the most important for interactions with RNA polymerase II.

"It's turned out to be extremely novel, revealing how a molecular machine is built from multiple proteins," said Takagi.

"As a molecular machine, the Mediator head module needs to have elements of both stability and flexibility in order to accommodate numerous interactions. A portion of the head we named the neck domain provides the stability by arranging the five proteins in a polymer-like structure," he said.



"We call it the alpha helical bundle," said Dr. Takagi. "People have seen structures of alpha helical bundles before but not coming from five different proteins."

A research team led by Yuichiro Takagi, Ph.D.,
Indiana University School of Medicine, has
deciphered the structure of an essential part
of Mediator, a complex molecular machine
that plays a vital role in regulating the
transcription of DNA.

"This is a completely noble structure," he said.

One immediate benefit of the research will be to provide detailed mapping of previously known mutations that affect the regulation of the transcription process, he said.

The ability to solve such complex structures will be important because multi-protein complexes such as Mediator will most likely become a new generation of drug targets for treatment of disease, he said.

Previously, the structure of RNA polymerase II was determined by Roger Kornberg of Stanford University, with whom Dr. Takagi worked prior to coming to IU School of Medicine. Kornberg received the Nobel Prize in 2006 for his discoveries. The researchers who described the structure of the ribosome, the protein production machine, were awarded the Nobel Prize in 2009. The structure of the entire Mediator has yet to be described, and thus remains the one of grand challenges in structure biology. Dr. Takagi's work on the Mediator head module structure represents a major step towards a structure determination of the entire Mediator. In addition to Dr. Takagi as a senior author, the lead author for the Nature paper was Tsuyoshi Imasaki, Ph.D., of the IU School of Medicine. Other collaborators included researchers at The Scripps Research Institute, Stanford University, Memorial Sloan-Kettering Cancer Center and the European Molecular Biology Laboratory.

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Your Body Recycling Itself -- Captured on Film

Your Body Recycling Itself -- Captured on Film

  8:26:00 PM    Science Lover

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Our bodies recycle proteins, the fundamental building blocks that enable cell growth and development. Proteins are made up of a chain of amino acids, and scientists have known since the 1980s that first one in the chain determines the lifetime of a protein. McGill researchers have finally discovered how the cell identifies this first amino acid -- and caught it on camera.

This image shows UBR-box recognition of an arginine residue at the beginning of a protein (blue) targeted for degradation. The structural integrity of the UBR box depends on zinc (grey) and a histidine residue (red) that is mutated in Johanson-Blizzard syndrome. (Credit: Department of Biochemistry, McGill University.)

"There are lots of reasons cells recycle proteins -- fasting, which causes loss of muscle, growth and remodeling during development, and normal turnover as old proteins are replaced to make new ones," explained lead researcher, Dr. Kalle Gehring, from McGill's Department of Biochemistry. "One way that cells decide which proteins to degrade is the presence of a signal known as an N-degron at the start of the protein. By X-ray crystallography, we discovered that the N-degron is recognized by the UBR box, a component of the cells' recycling system."

The powerful technique can pinpoint the exact location of atoms and enabled the team to capture an image of the UBR box, providing insight to this incredibly tiny yet essential part of our bodies' chemical mechanics.

Aside from representing a major advance in our understanding of the life cycle of proteins, the research has important repercussions for Johanson-Blizzard syndrome, a rare disease that causes deformations and mental retardation. This syndrome is caused by a mutation in the UBR box that causes it to lose an essential zinc atom. Better understanding of the structure of the UBR box may help researchers develop treatments for this syndrome.

The research was published in Nature Structural & Molecular Biology and received funding from the Canadian Institutes of Health Research.

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Study Reveals Structure of Cell Division’s Key Molecule At the Crossroads of Chromosomes

Study Reveals Structure of Cell Division’s Key Molecule At the Crossroads of Chromosomes

  1:25:00 AM    Science Lover

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On average, one hundred billion cells in the human body divide over the course of a day. Most of the time the body gets it right but sometimes, problems in cell replication can lead to abnormalities in chromosomes resulting in many types of disorders, from cancer to Down Syndrome.

Human chromosome, with conventional nucleosomes 
containing the major form of the histones (green), and 
localization of the centromere histone H3 variant, 
CENP-A (red). (Credit: Ben E. Black, University 
of Pennsylvania School of Medicine)

Now, researchers at the University of Pennsylvania's School of Medicine have defined the structure of a key molecule that plays a central role in how DNA is duplicated and then moved correctly and equally into two daughter cells to produce two exact copies of the mother cell. Without this molecule, entire chromosomes could be lost during cell division.

Ben Black, PhD, assistant professor of Biochemistry and Biophysics, and Nikolina Sekulic, PhD, a postdoctoral fellow in the Black lab, report in the Sept. 16 issue of Nature the structure of the CENP-A molecule, which defines a part of the chromosome called the centromere. This is a constricted area to which specialized molecules called spindle fibers attach that help pull daughter cells apart during cell division.

"Our work gives us the first high-resolution view of the molecules that control genetic inheritance at cell division," says Black. "This is a big step forward in a puzzle that biologists have been chipping away at for over 150 years."

Investigators have known for the last 15 years that part of cell division is controlled by epigenetic processes, the series of actions that affect the protein spools around which DNA is tightly bound, rather than encoded in the DNA sequence itself. Those spools are built of histone proteins, and chemical changes to these spool proteins can either loosen or tighten their interaction with DNA. Epigenetics alter the readout of the genetic code, in some cases ramping a gene's expression up or down. In the case of the centromere, it marks the site where spindle fibers attach independently of the underlying DNA sequence. CENP-A has been suspected to be the key epigenetic marker protein.

However, what hasn't been known is how CENP-A epigenetically marks the centromere to direct inheritance. The Black team found the structural features that confer CENP-A the ability to mark centromere location on each chromosome. This is important because without CENP-A or the centromere mark it creates, the entire chromosome -- and all of the genes it houses -- are lost at cell division.

In this study, Black solved CENP-A's structure to determine how it specifically marks the centromere on each chromosome and surmise from that how the epigenetic mark is copied correctly in each cell division. They found that CENP-A changes the shape of the nucleosome of which it's a part, also making it more rigid than other nucleosomes without CENP-A. The nucleosome is the combination of DNA wound around a histone protein core --the DNA thread wrapped around the histone spool. The CENP-A nucleosome is copied several times to create a unique epigenetic area, different from the rest of the chromosome. CENP-A replaces histone H3 in the nucleosomes located at the centromere.

This CENP-A centromere identifier attracts other proteins, and in cell division builds a massive structure, the kinetochore, for pulling the duplicated chromosomes apart during cell division.

Besides the major advance in the understanding of the molecules driving human inheritance, this work also brings about the exciting prospect that the key epigenetic components are now in hand to engineer clinically useful artificial chromosomes that will be inherited alongside our own natural chromosomes -- and with the same high fidelity, says Black.

Co-authors are graduate student Emily A. Bassett and research specialist Danielle J. Rogers. The work was funded by National Institute for General Medical Sciences, the Burroughs Wellcome Fund, the Rita Allen Foundation, the American Cancer Society, and the American Heart Association.

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RNA Network Seen in Live Bacterial Cells for First Time

RNA Network Seen in Live Bacterial Cells for First Time

  12:49:00 AM    Science Lover

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Scientists who study RNA have faced a formidable roadblock: trying to examine RNA's movements in a living cell when they can't see the RNA. Now, a new technology has given scientists the first look ever at RNA in a live bacteria cell -- a sight that could offer new information about how the molecule moves and works.

These are fluorescent images of E. coli bacterial cells with visualized RNA. The bar denotes 2 microns. (Credit: Image courtesy of Natalia E. Broude, Ph.D. / Department of Biomedical Engineering, Boston University)

Interest in RNA, which plays a key role in manufacturing proteins, has increased in recent years, due in large part to its potential in new drug therapies. RNA localization and movement in bacterial cell are poorly understood. The problem has been finding a way to mark RNA in a living cell so that scientists can track it, says Natasha Broude, a research associate professor at Boston University's Department of Biomedical Engineering.

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Nanotechnology: Artificial Pore Created

Nanotechnology: Artificial Pore Created

  12:35:00 AM    Science Lover

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Using an RNA-powered nanomotor, University of Cincinnati (UC) biomedical engineering researchers have successfully developed an artificial pore able to transmit nanoscale material through a membrane.

Scientists inserted the modified core of a nanomotor, a microscopic biological machine, into a lipid membrane. The resulting channel enabled them to move both single- and double-stranded DNA through the membrane. (Credit: Image courtesy of University of Cincinnati)

In a study led by UC biomedical engineering professor Peixuan Guo, PhD, members of the UC team inserted the modified core of a nanomotor, a microscopic biological machine, into a lipid membrane. The resulting channel enabled them to move both single- and double-stranded DNA through the membrane.

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