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Posts Tagged ‘rna’

How cell’s master transcribing machine achieves near perfection

One of the most critical processes in biology is the transcription of genetic information from DNA to messenger RNA (mRNA), which provides the blueprint for the proteins that form the machinery of life. Now, researchers have discovered new details of how the cell’s major transcriptional machinery, RNA polymerase II (Pol II), functions with such exquisite precision. With almost unerring accuracy, Pol II can select the correct molecular puzzle piece, called a nucleosidetriphosphate (NTP), to add to the growing mRNA chain, although these puzzle pieces can be highly similar molecules.

Two papers in the June 6, 2008, issue of the journal Molecular Cell, published by Cell Press, describe advances in understanding Pol II copying fidelity. The papers are by Craig Kaplan of Stanford University and his colleagues; and Mikhail Kashlev of the National Cancer Institute Center for Cancer Research and his colleagues.

The researchers said their findings not only offer unprecedented details about the fidelity mechanism of Pol II, but likely about fidelity in all cellular genetic copying machines. They said their discoveries also offer understanding of how defective Pol II can generate errors in transcribing mRNA—errors that can promote cancer formation.
Both groups concentrated on the function of the Pol II “active site” region, where the enzyme captures an RNA component, called a nucleosidetriphosphate (NTP), and chemically attaches it to the RNA chain. Although Pol II uses the DNA genetic sequence as a template to specify the RNA sequence, another largely unknown fidelity mechanism exists by which Pol II discriminates against incorrect NTPs. This fidelity mechanism is extremely precise; it can distinguish the NTPs that make up RNA from the deoxyNTPs used in DNA—although the two molecules differ only in one small chemical group.

In their paper, Kaplan and colleagues explored a key component of the active site known as the “trigger loop.” This small bit of protein is highly mobile, and although researchers have believed that it plays a critical function in discriminating the correct NTP, that function was poorly understood.

In studies with yeast, Kaplan and his colleagues produced a mutant form of Pol II with a subtly crippled trigger loop. This mutation substituted one amino acid with another in what was believed to be a key position in the trigger loop, His 1085, for interacting with incoming NTPs to discriminate the correct one. The researchers compared the detailed molecular function of normal and His 1085 mutant Pol II enzymes during the encounter with both correct and incorrect NTPs. They also compared the behavior of the mutant with the action of the mushroom toxin alpha-amanitin, which is theorized to block Pol II by interfering with the trigger loop. The researchers’ studies of the mutant and alpha-amanitin revealed crucial details showing how the trigger loop determines fidelity, said Kaplan.

“We found that the amanitin-treated wild-type enzyme behaved very similar to our mutant enzyme,” said Kaplan. In fact, he said, the experiments, as well as structural information on the active site, indicated that alpha-amanitin targets the same His 1085 position in the trigger loop as does their mutation. Kaplan concluded that the findings reveal a specific and critical role for the trigger loop.

“These findings reveal what is called a ‘kinetic selection’ mechanism for Pol II, which is like many polymerases,” he said. “That is, the active site in one condition has a similar affinity for both correct and incorrect NTPs. However, because of motion within the active site—in this case the action of the trigger loop—catalytic activity in the active site proceeds much faster with the correct NTP than with the incorrect NTP. The trigger loop is mobile, and only when it is positioned properly in response to a correct substrate can it really function.

“We think this mode of substrate recognition is a general theme for systems that have to select the right molecule out of a giant pool of the wrong molecules,” said Kaplan. An example, he said, is when the protein-making ribosomal machinery must select the correct transfer RNA from among similar-but-incorrect transfer RNAs.

Besides Kaplan, other co-authors on the paper were Karl-Magnus Larsson and Roger Kornberg.

In the other Molecular Cell paper, Kashlev and colleagues used a different yeast mutant to explore the function of the Pol II active site. In their screen for Pol II mutants, they identified one, E1103G, that shows a several-fold increase in error rate over the normal, wild-type Pol II.

Importantly, said Kashlev, the researchers could precisely measure the transcription error rate using a new assay, called a retrotransposition assay, developed by co-author Jeffrey Strathern.

The researchers’ analysis of the effects of E1103G yielded significant insights into the function of the trigger loop, said Kashlev.

“Normally, when an NTP diffuses into the active site of the polymerase, the trigger loop closes behind it like a door, long enough for the polymerase to perform the chemistry to add the NTP to the end of the RNA chain,” he said. “If the NTP is incorrect, there is a tendency for this door to stay open for a longer time, which means that the NTP has a chance to diffuse out of the active site before the polymerase can proceed to chemistry.

“Our mutation occupies a strategic position important for keeping the loop open, like a latch,” said Kashlev. “So, in the mutant, the door wants to stay in the closed state for a longer time, which means if an incorrect NTP migrates into the active site, there is time for the polymerase to add this incorrect NTP to the RNA chain.”

Kashlev said the motivation for their studies of Pol II transcription fidelity is to understand the effects of Pol II errors on genome stability. Specifically, error-prone Pol II could generate mRNA that produces aberrant versions of the critical enzyme DNA polymerase. As DNA polymerase is responsible for gene replication, the result of its malfunction could be a burst of gene mutation causing an “error catastrophe” that could lead to genome instability and cancer formation.

Source: Cell Press

The RNA Polymerase II Trigger Loop Functions in Substrate Selection and Is Directly Targeted by α-Amanitin. Craig D. Kaplan, Karl-Magnus Larsson, and Roger D. Kornberg. Molecular Cell. June 5, 2008: 30 (5).

Transient Reversal of RNA Polymerase II Active Site Closing Controls Fidelity of Transcription Elongation. Maria L. Kireeva, Yuri A. Nedialkov, Gina H. Cremona, Yuri A. Purtov, Lucyna Lubkowska, Francisco Malagon, Zachary F. Burton, Jeffrey N. Strathern, and Mikhail Kashlev. Molecular Cell. June 5, 2008: 30 (5)

Josh says:

Perhaps it’s just because I had a class that focused primarily on DNA replication and RNA transcription, but I find this fascinating. I immediately recognized that the one paper came from Roger Kornberg‘s lab. This also reminds me of a video from The Walter and Eliza Hall Institute of Medical Research (WEHI) of DNA transcription into RNA. More videos can be found at their site. Sorry for linking to a quicktime movie, but youtube (nor Linux) will play the Quicktime movie correctly, and wordpress won’t let me embed a quicktime movie

Andrew says:

Somebody from Reddit in the comments suggested we post this video: How Cell Achieves Perfection

RNA toxicity contributes to neurodegenerative disease, University of Pennsylvania scientists say

Expanding on prior research performed at the University of Pennsylvania, Penn biologists have determined that faulty RNA, the blueprint that creates mutated, toxic proteins, contributes to a family of neurodegenerative disorders in humans.

Nancy Bonini, professor in the Department of Biology at Penn and an investigator of the Howard Hughes Medical Institute, and her team previously showed that the gene that codes for the ataxin-3 protein, responsible for the inherited neurodegenerative disorder Spinocerebellar ataxia type 3, or SCA3, can cause the disease in the model organism Drosophila. SCA3 is one of a class of human diseases known as polyglutamine repeat diseases, which includes Huntington’s disease. Previous studies had suggested that the disease is caused largely by the toxic polyglutamine protein encoded by the gene.

The current study, which appears in the journal Nature, demonstrates that faulty RNA, the blueprint for the toxic polyglutamine protein, also assists in the onset and progression of disease in fruit fly models.

“The challenge for many researchers is coupling the power of a simple genetic model, in this case the fruit fly, to the enormous problem of human neurodegenerative disease,” Bonini said. “By recreating in the fly various human diseases, we have found that, while the mutated protein is a toxic entity, toxicity is also going on at the RNA level to contribute to the disease.”

To identify potential contributors to ataxin-3 pathogenesis, Bonini and her team performed a genetic screen with the fruit fly model of ataxin-3 to find genes that could change the toxicity. The study produced one new gene that dramatically enhanced neurodegeneration. Molecular analysis showed that the gene affected was muscleblind, a gene previously implicated as a modifier of toxicity in a different class of human disease due to a toxic RNA. These results suggested the possibility that RNA toxicity may also occur in the polyglutamine disease situation.

The findings indicated that an RNA containing a long CAG repeat, which encodes the polyglutamine stretch in the toxic polyglutamine protein, may contribute to neurodegeneration beyond being the blueprint for that protein. This raised the possibility that expression of the RNA alone may be damaging.

Long CAG repeat sequences can bind together to form hairpins, dangerous molecular shapes. The researchers therefore tested the role of the RNA by altering the CAG repeat sequence to be an interrupted CAACAG repeat that could no longer form a hairpin. Such an RNA strand, however, would still be a blueprint for an identical protein. The researchers found that this altered gene caused dramatically reduced neurodegeneration, indicating that altering the RNA structure mitigated toxicity. To further implicate the RNA in the disease progression, the researchers then expressed just a toxic RNA alone, one that was unable to code for a protein at all. This also caused neuronal degeneration. These findings revealed a toxic role for the RNA in polyglutamine disease, highlighting common components between different types of human triplet repeat expansion diseases. Such diseases include not only the polyglutamine diseases but also diseases like myotonic dystrophy and fragile X.

The family of diseases called polyglutamine repeat disorders arise when the genetic code of a CAG repeat for the amino acid glutamine stutters like a broken record within the gene, becoming very long. This leads to an RNA — the blueprint for the protein — with a similar long run of CAG. During protein synthesis, the long run of CAG repeats are translated into a long uninterrupted run of glutamine residues, forming what is known as a polyglutamine tract. The expanded polyglutamine tract causes the errant protein to fold improperly, leading to a glut of misfolded protein collecting in cells of the nervous system, much like what occurs in Alzheimer’s and Parkinson’s diseases.

Polyglutamine disorders are genetically inherited ataxias, neurodegenerative disorders marked by a gradual decay of muscle coordination, typically appearing in adulthood. They are progressive diseases, with a correlation between the number of CAG repeats within the gene, the severity of disease and age at onset.

Source: University of Pennsylvania

RNA toxicity is a component of ataxin-3 degeneration in Drosophila. Ling-Bo Li, Zhenming Yu, Xiuyin Teng & Nancy M. Bonini. Nature (2008). doi:10.1038/nature06909

Josh says:

I don’t think anyone has looked at this before or considered that RNA could have such an effect. I wonder how much this type of thing affects other diseases, both neurological and non-neurological.

Andrew says:

So because RNA is a molecule, not just an “abstract informational template” as we often think, the physical manifestation of RNA’s information can a significant impact on the biological system —not just the information itself! In this case, the issue is the “hairpin” shape of a CAG string in RNA. So again, the metaphor “genes as code” fails. Fascinating.

Analysis of RNA role in spreading disease advances study of damaging plant infections

Recent research that links specific pieces of RNA to an infectious organism’s duplication and spread could lead the way to the prevention of viroids, pathogens that can kill or damage food crops and other plants.

The findings and the research approach used by Ohio State University scientists also could have applications in the study of how certain viruses spread in humans because the pathogens have some similar characteristics. … Continue Reading »

New research shows promising gene therapy technique may have harmful side effects

A dramatic new study published in the most recent issue of Nature questions some of the mechanisms underlying a new class of drugs based on Nobel Prize-winning work designed to fight diseases ranging from macular degeneration to diabetes.

Dr. Jayakrishna Ambati, a University of Kentucky researcher and the paper’s senior author, has for years been investigating gene silencing, a 1998 discovery that won a Nobel Prize in Physiology or Medicine in unusually quick fashion in 2006.

While the prize-winning discovery remains important, the findings made by Ambati’s lab show the mechanisms behind it are not as scientists once believed. In fact, Ambati’s work imparts the need for caution in current clinical trials using the technology, as it may have potentially harmful effects on subjects. … Continue Reading »