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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Environs Prompt Advantageous Gene Mutations as Plants Grow; Changes Passed to Progeny

Environs Prompt Advantageous Gene Mutations as Plants Grow; Changes Passed to Progeny

  1:21:00 PM    Science Lover

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If a person were to climb a towering redwood and take a sample from the top and a sample from the bottom of the tree, a comparison would show that the two DNA samples are different.

Research shows that if a person were to climb a towering redwood and take a sample from the top and a sample from the bottom of the tree, a comparison would show that the two DNA samples are different. (Credit: © Galyna Andrushko / Fotolia)

Christopher A. Cullis, chair of biology at Case Western Reserve University, explains that this is the basis of his controversial research findings.

Cullis, who has spent over 40 years studying mutations within plants, most recently flax (Linum usitatissimum), has found that the environment not only weeds out harmful and useless mutations through natural selection, but actually influences helpful mutations.

Cullis published his findings in the International Journal of Genetics and Molecular Biology and repeated them in the Journal of Visualized Experiments, where he challenged other scientists to repeat his experiment themselves.

Specifically, Cullis focuses on mutations involving the appearance of a small sequence of DNA known as LIS-1 and how the environment affects these changes.

The controversy stems from the idea that the environment changes organisms as they grow and these changes are passed on.

While originally accepted, the theory was eventually thrown out because science revealed that animals pass along DNA through their gamete or sex cells, which are not affected by the environment. This concept was assumed to be the same for plants, but Cullis's research says otherwise.

In his second study, three separate strands (the plastic strand, short strand, and tall strand) of the Stormont Cirrus variety of flax were grown under three separate conditions.

Each of the strands had been bred over multiple generations under different conditions: The plastic strand's ancestors were grown under control conditions, the short strand's ancestors were grown under low-nutrient conditions, and the tall strand's ancestors were grown under high-nutrient conditions.



The experiment showed each strand responded to each condition in a different way, corresponding to the environment its ancestors were grown in. The plastic strand outgrew the other strands under control conditions, the short strand outgrew the other strands when few nutrients were available, and the tall strand grew best when nutrients were readily available.

All this information does not completely explain Cullis's assertion that the environment can in a single generation help sift out the useful mutations.

This is where polymerase chain reaction (PCR) amplification of DNA comes in. Through this process, the researchers could see when a specific DNA sequence (in this case LIS-1) appears or disappears.

When the plastic strand is grown under low nutrient conditions, the LIS-1 sequence, which had been absent, appears and continues for future generations. Since the LIS-1 sequence helps plants survive when there is a shortage of nutrients, its presence helps confirm Cullis's belief that the environment can act on how a plant mutates and keep helpful mutations, even within one generation.

These findings help explain why the top of a redwood is genetically different from the bottom. Young redwoods grow by the tips of the existing branches budding into meristems. Each new meristem is different from the tree because the environment has affected its genetic makeup. And as the redwood grows, the top becomes more and more genetically different from the bottom.

Due to the controversy surrounding Cullis's findings, many scientists are hesitant to accept them as true. Cullis himself recalls at first being skeptical and thinking, "If this really works… [we can] get a plant that's better adapted to its environment in one generation."

These adapted plants have practical uses. Cullis hopes to identify the specific gene sequence responsible for flax's ability to withstand harsh environments and insert it into the DNA sequence of other plants so that they too can withstand trying environments.

This would bypass the current method of genetically engineering plants, which involves isolating specific DNA sequences that control heat-resistance, cold-resistance, pest-resistance, etc., and instead narrows the effort down to one DNA sequence.

By inserting this sequence into the plant and growing it in a specific trying environment, scientists could make the plant resistant to what they want. All of that plant's offspring would be adapted to the environment and ready to grow.

By making the plant do all the work, the price of producing better crops would be greatly reduced. This would greatly benefit developing nations that need a large supply of food in an otherwise harsh environment. The DNA sequence may no longer just help the plant survive, but can now help entire countries thrive.

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Human Genome's Breaking Points: Genetic Sequence of Large-Scale Differences Between Human Genomes

Human Genome's Breaking Points: Genetic Sequence of Large-Scale Differences Between Human Genomes

  10:04:00 AM    Science Lover

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A detailed analysis of data from 185 human genomes sequenced in the course of the 1000 Genomes Project, by scientists at the European Molecular Biology Laboratory (EMBL) in Heidelberg, Germany, in collaboration with researchers at the Wellcome Trust Sanger Institute in Cambridge, UK, as well as the University of Washington and Harvard Medical School, both in the USA, has identified the genetic sequence of an unprecedented 28 000 structural variants (SVs) -- large portions of the human genome which differ from one person to another.

The work, published in Nature, could help find the genetic causes of some diseases and also begins to explain why certain parts of the human genome change more than others.

Scientists have identified the genetic sequence of an unprecedented 28,000 structural variants -- large portions of the human genome which differ from one person to another. (Credit: iStockphoto/Andrey Prokhorov)

The international team of scientists identified over a thousand SVs that disrupt the sequence of one or more genes. These gene-altering mutations may be linked to diseases, so knowing the exact genetic sequence of these variations will help clinical geneticists to narrow down their searches for disease-causing mutations.

"Knowing the exact genetic sequence of SVs and their context in the genome could help find the genetic causes for as-yet unexplained diseases," says Jan Korbel, who led the research at EMBL: "this may help us understand why some people remain healthy until old age whereas others develop diseases early in their lives."

This unprecedented catalogue of large-scale genetic variants also sheds light on why some parts of the genome mutate more frequently than others. The scientists found that deletions, where genetic material is lost, and insertions, where it is gained, tend to happen in different places in the genome and through different molecular processes. For instance, large-scale deletions are more likely to occur in regions where DNA often breaks and has to be put back together, as 'chunks' of genetic material can be lost in the process.

"We found 51 hotspots where certain SVs, such as large deletions, appear to occur particularly often" Korbel says: "Six of those hotspots are in regions known to be related to genetic conditions such as Miller-Dieker syndrome, a congenital brain disease that can lead to infant death."

Previous research had already linked SVs -- also called copy-number variants -- to many genetic conditions, such as colour-blindness, schizophrenia, and certain forms of cancer. However, because of their large size and complex DNA sequence, SVs were difficult to identify. In this study, the researchers overcame these difficulties, developing novel computational approaches that allowed them to pinpoint the exact locations of these large-scale variations in the genome, broadening the potential scope of future disease studies.

"There are many structural variants in everyone's genomes and they are increasingly being associated with various aspects of human health" says Charles Lee, a clinical cytogeneticist and associate professor at Harvard Medical School and Brigham and Women's Hospital, and joint leader of the study: "It is important to be able to identify and comprehensively characterize these genetic variants using state-of-the-art DNA sequencing technologies."

Data from this study is being made publicly available to the scientific community through the 1000 Genomes Project, an international public-private consortium to build the most detailed map of human genetic variation to date. The 1000 Genomes Project aims to sequence 2500 whole genomes by the end of 2012, resulting, by far, in the largest collection of human genomes to date.

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