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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Non-coding RNA has role in inherited neurological disorder -- and maybe other brain diseases too

Non-coding RNA has role in inherited neurological disorder -- and maybe other brain diseases too

  1:26:00 AM    Science Lover

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A team of scientists, led by researchers at the University of California, San Diego School of Medicine, have uncovered a novel mechanism regulating gene expression and transcription linked to Spinocerebellar ataxia 7, an inherited neurological disorder. The discovery promises to have broad ramifications, suggesting that abundant non-coding transcripts of ribonucleic acid (RNA) may be key players in neurological development and function, and could be powerful targets for future clinical therapies.

Researchers have discovered that expression
of the ataxin‑7 gene - the cause of the
neurological disorder spinocerebellar
ataxia type 7 - has two regulators: a highly
conserved, multi‑tasking protein called
CTCF and, surprisingly, an adjacent promoter
containing non‑coding RNA.
Credit: Illustration courtesy of Christina
Takamatsu‑Butler, UC San Diego.

The research, headed by Albert La Spada, MD, PhD, chief of the division of genetics in the UCSD department of pediatrics, and professor of cellular and molecular medicine, neurosciences and biological sciences, is published in the June 22 issue of the journal Neuron.

"Our paper highlights a number of important emerging themes in our understanding of gene regulation in the brain," said La Spada, who is also associate director of the UCSD Institute for Genomic Medicine.

"With the advent of new technologies, science has learned that the vast majority of our transcripts are non-coding," said La Spada. "The challenge going forward is to determine what they do do, and if they have specific functions. It now seems increasingly likely that a multitude of these non-coding RNAs help finely tune transcription regulation in the brain, and perturbation of their work is linked to disease. If we can figure out exactly how, we should be able to gain new insights into how the brain is so precisely regulated – knowledge that may help us better understand how the brain works."



Spinocerebellar ataxia 7 is one of several types of spinocerebellar ataxia (SCA), genetic degenerative disorders characterized by atrophy in the cerebellum of the brain, progressive loss of physical coordination – and in the case of type 7 – retinal degeneration that can result in blindness. There is currently no known cure.

Many SCAs are classified as polyglutamine diseases, caused when a protein associated with the disease contains too many repeats of the amino acid glutamine. Polyglutamine diseases are also known as "CAG Triplet Repeat Disorders" because CAG is the sequence of nucleic acids that codes for glutamine.

La Spada and colleagues have long studied SCA. In 2001, they were the first to demonstrate that SCA7 retinal degeneration was the result of transcription dysregulation of ataxin-7, the protein associated with SCA7. Following up, they decided to learn how the gene that expresses ataxin-7 is itself regulated.

The researchers found not one, but two, regulators. The first is called CTCF, a highly conserved protein that regulates a variety of transcriptional processes, most notable establishing insulator domains and controlling genomic imprinting. But they also discovered an adjacent, alternative promoter dubbed intron 2 promoter (P2A) and a transcribed antisense, non-coding RNA, which they labeled SpinoCerebellarAtaxia-AntisenseNoncodingTranscript1 or SCAANT1.

Antisense RNA is single-stranded ribonucleic acid whose primary function appears to be as an inhibitor or suppressor of a gene, though sometimes it can promote gene expression instead. Most antisense RNAs are non-coding, meaning that their sequences do not provide information for making proteins. Even though non-coding RNAs do not provide instructions for the production of vital proteins, they comprise the bulk of the human genome. A major challenge for biomedical research in the 21st century is to figure what they do, and how they do it.

In their Neuron paper, La Spada and colleagues highlight one function, at least for SCAANT1. When they investigated how CTCF regulated ataxin-7 gene expression in transgenic mice, they discovered that CTCF promotes the production of SCAANT1 which in turn represses the newly discovered ataxin-7 sense promoter P2A. In mice lacking SCAANT1, sense promoter P2A is de-repressed, allowing a mutant ataxin-7 gene to be expressed, resulting in mice with a version of SCA7. The scientists found a similar lack of antisense SCAANT1 in the fibroblasts and white blood cells taken from human patients with SCA7, implicating deregulation of this pathway in the disease process.

As many inherited neurological disorders are now known to exhibit such overlapping "bidirectional" transcription, the findings in SCA7 could shed light on similar abnormalities with non-coding RNA function in a number of brain diseases.

Provided by University of California - San Diego

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Scientists Override Errant Form of Genetic Signaling for First Time: Changing Genetic 'Red Light' to Green Holds Promise for Treating Disease

Scientists Override Errant Form of Genetic Signaling for First Time: Changing Genetic 'Red Light' to Green Holds Promise for Treating Disease

  3:24:00 PM    Science Lover

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In a new study published June 15 in the journal Nature, scientists discovered an entirely new way to change the genetic code. The findings, though early, are significant because they may ultimately help researchers alter the course of devastating genetic disorders, such as cystic fibrosis, muscular dystrophy and many forms of cancer.

Scientists discovered an entirely new way to change 
the genetic code. (Credit: © Rodolfo Clix / Fotolia)

The genetic code is the set of instructions in a gene that tell a cell how to make a specific protein. Central to the body's protein production process is messenger RNA, or mRNA, which takes these instructions from DNA and directs the steps necessary to build a protein. For the first time, researchers artificially modified messenger RNA, and in doing so changed the original instructions for creating the protein. The end result: A different protein than originally called for.

"The ability to manipulate the production of a protein from a particular gene is the new miracle of modern medicine," said Robert Bambara, Ph.D., chair of the Department of Biochemistry and Biophysics at the University of Rochester Medical Center. "This is a really powerful concept that can be used to try to suppress the tendency of individuals to get certain debilitating, and sometimes fatal genetic diseases that will forever change their lives."

Protein production is not a perfect process -- far from it. Frequent mutations or mistakes in DNA and messenger RNA can lead to flawed proteins that have the potential to cause serious harm. In the study, researchers focused on a common type of mutation that occurs when an mRNA molecule contains a pre-mature "stop" signal, known as a pre-mature stop codon. A premature stop codon orders a cell to stop reading the genetic instructions partway through the process, resulting in the creation of an incomplete, shortened protein.



Researchers were able to alter mRNA in a way that turned a stop signal into a "go" signal. As a result, the cell could read the genetic instructions all the way through and create a normal, full-length protein. The team produced these results both in vitro and in live yeast cells.

"This is a very exciting finding," said Yi-Tao Yu, Ph.D., lead study author and associate professor of Biochemistry and Biophysics at the Medical Center. "No one ever imagined that you could alter a stop codon the way we have and allow translation to continue uninterrupted like it was never there in the first place."

The findings are important because current estimates suggest that approximately one third of genetic diseases are caused by the presence of pre-mature stop codons that result in shortened proteins. The results could aid the development of treatment strategies designed to help the body override stop codons and produce adequate amounts of full-length proteins, whose absence causes diseases like cystic fibrosis and contributes to different types of cancer.

Yu, along with first author John Karijolich, Ph.D., used another type of RNA -- guide RNA -- to modify messenger RNA. Guide RNAs are short RNAs that bind to specific sequences in RNA and allow just one particular site to be modified. "Guide RNAs give us tremendous power to zero in on one spot in the genome and make very targeted changes," noted Bambara.

The team developed an artificial guide RNA and programmed it to target and change a specific stop codon in an mRNA.

"The fact that this strategy worked -- that the guide RNA we created found its way to its target, the stop codon, and directed the desired structure change -- is pretty remarkable. Guide RNAs weren't thought to have access to messenger RNA, so no one believed they could target messenger RNA for modification," said Karijolich, who conducted the research as a graduate student at Rochester, but is now a postdoctoral fellow in the Department of Biochemistry at the Robert Wood Johnson Medical School. "Our results bring up the question of whether a similar process may be happening naturally."

"Previous research has presented other ways to modify the genetic code, but what is really unique about our method is that it is at the RNA level and it is site specific. We can express the artificial guide RNA in a cell and direct it to make a modification at a single site and only that site," said Yu.

Altering messenger RNA in this way may be another mechanism human cells use to create many different types of proteins. Given our complexity, humans have surprisingly few genes. While it is well established that the majority of human genes code for more than one protein, mRNA modification may be an unrealized way that humans are able to do this.

Yu plans to pursue this research further, studying whether and how targeted mRNA modification is happening naturally.

The study was funded by the National Institute of General Medical Sciences at the National Institutes of Health.

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A New Way to Make Stem Cells Using RNA instead of DNA could avoid the health risks--and the political pitfalls--of stem-cell treatments.

A New Way to Make Stem Cells Using RNA instead of DNA could avoid the health risks--and the political pitfalls--of stem-cell treatments.

  1:27:00 AM    Science Lover

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A Harvard researcher has developed a way to make pluripotent stem cells that solves several of the major impediments to using them to treat human diseases.

New cell: This microscope image shows neurons
(colored green) created from pluripotent stem
cells using modified RNA.
Credit: Phil Manos, Cell Press

Derrick Rossi, an assistant professor at Harvard Medical School, created pluripotent stem cells--which can turn into virtually any other type of cell in the body--from non-stem cells without using viruses to tinker with a cell's genome, as conventional methods do. This means that Rossi's method could be substantially safer for treating disease. The work is published today in the journal Cell Stem Cell.

"Rossi has figured out how to turn a skin cell into a stem cell without genetic modifications, and to do it efficiently," said Doug Melton, codirector of the Harvard Stem Cell Institute, where Rossi is a principal faculty member, at a press conference.

Rossi's innovation, which has not yet been tested in people, was to use messenger RNA instead of DNA to produce the four proteins needed to reprogram the cell. He has started a company called ModeRNA to commercialize this use of messenger RNA. He said the approach may also have potential in gene therapy, which also relies on viruses to deliver treatment, but he declined to talk further about the company or possible gene therapy applications because the work is at such an early stage.

Improving the usability of man-made stem cells is key to helping patients and ending the political morass that has slowed stem-cell research. On Tuesday, a U.S. federal appeals court allowed federal funding of embryonic stem-cell research to continue while a legal case against such funding proceeds.

The human embryonic stem cells used in research were mostly derived from embryonic tissue grown a decade ago. These are the most versatile cells in the body, and the gold standard by which man-made cells are judged.

Four years ago, Japanese researcher Shinya Yamanaka showed that regular cells could be turned into embryonic-like stem cells--called induced pluripotent stem (iPS) cells--through the introduction of four specific proteins. Theoretically, this meant that doctors could take skin cells from a sick or disabled person, transform them into stem cells, and then into a specialized cell to treat them--an insulin-producing islet cell for someone with diabetes or a nerve cell for someone who is paralyzed, for instance. Using iPS cells avoids the need to destroy embryos and, because they can be derived from the patient's own cells, means less risk of rejection.

But only one in 1,000 or one in 10,000 skin cells could be transformed into a stem cell using Yamanaka's method. It also changes a cell's genes in ways that might trigger cancer or other problems.

Rossi 's idea was to produce Yamanaka's four proteins in a different way. Instead of using the DNA that holds the instructions for making proteins, he wanted to use RNA, which carries those instructions to the place in a cell where proteins are made.

His first several attempts were miserable failures. When he tried to change the messages the RNA carried, he triggered a serious immune response and most of the cells shut down or self-destructed. Rossi then tried modifying the RNA chemically and eventually figured out a way to allow his changes to escape immune detection while delivering the message. "This was key to our success," said Rossi, who is also a researcher at Children's Hospital Boston. "We could encode RNA for any protein we wanted to express and insert it into a cell."

Rossi said it was a happy coincidence that using RNA instead of changing the DNA was as much as 100-fold more efficient. He said the effect was possibly because the process more closely reflects how cells themselves transform.

Rossi successfully differentiated his stem cells into muscle cells using RNA, a process that may offer promise in gene therapy and other treatments. His method does not alter the cell's underlying genome, though Rossi admits that he does not yet understand what it does to the cell's epigenome, which controls expression of genes.

Rossi said that his cells, which he's named RiPS, for "RNA induced Pluripotent Stem" cells, are more like embryonic stem cells than traditional iPS cells because they have not been genetically altered.

Melton said the Harvard Stem Cell Institute, which includes several hundred stem-cell researchers from across Harvard University and its affiliated hospitals, will now be making its standard iPS cells with Rossi's method.

In a prepared statement, Yamanaka, now at the University of California, San Francisco, said Rossi's approach to generating stem cells seems promising, and he would like to have someone in his lab try it.

"The quality of the induced pluripotent stem cells generated by this method should be carefully examined because their characteristics vary depending on the induction methods and the origins of the resulting cells," he said. "The standard method to generate iPSCs for clinical applications has yet to be established. I think this method has the potential for it."

Jacob Hanna, a postdoctoral fellow at the Whitehead Institute for Biomedical Research in Cambridge, said he's also eager to begin working with the cells.

"I think it's a very exciting paper with a very promising method," said Hanna, who was not involved in the research. When asked if he was jealous that Rossi had developed the method first, Hanna said, "Yes, of course! It's a very nice paper," quickly adding, "Jealous in a very positive and supporting way."

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