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Posts Tagged ‘DNA’

Scientists discover DNA knot keeps viral genes tightly corked inside shell

A novel twist of DNA may keep viral genes tightly wound within a capsule, waiting for ejection into a host, a high-resolution analysis of its structure has revealed.

A donut-shaped twist, or toroid, of DNA (shown in red) wedges the viral genome tightly within the protein envelope of the bacteriophage.Using electron microscopy and three-dimensional computer reconstruction, UC San Diego biologists and chemists have produced the most detailed image yet of the protein envelope of an asymmetrical virus and the viral DNA packed within, they report this week in the journal Structure. The image, with a resolution of less than a nanometer, or a millionth of a millimeter, will help to unravel how the virus locks onto its host and infects the cells by injecting its DNA.

By assembling more than 12,000 microscopic views of frozen viral particles from different angles, UCSD chemists Jinghua Tang, Norman Olson and Timothy Baker, a professor of chemistry and biological sciences, have determined the structure of a bacteriophage called phi29 with a resolution finer than 8 Angstroms (one Angstrom equals a tenth of a nanometer). Their project was part of a long-term collaboration with molecular virologist Dwight Anderson and his colleagues at the University of Minnesota.

Although the structures of spherical viruses with a high degree of symmetry have been resolved using similar methods, many more images were required to accomplish the same task for the head-and-tail shape of phi29. The UCSD scientists said their images of phi29 are twice as fine as those created in previous efforts to visualize viruses with a similar shape.

A comparison between images of the virus with and without its DNA cargo revealed that the DNA twists tightly into a donut shape, or toroid, in the neck of the virus between its head and tail. “This highly distorted DNA structure is unlike anything previously seen or even predicted in a virus,” said Timothy Baker who headed the research team. “It’s an improbably tight turn for DNA, which is generally considered inflexible over very small distances.”

During assembly of the virus, a molecular motor in the neck winds the DNA strand into a tight coil within the head. “It’s under tremendous pressure — about 20 times that of champagne in a bottle,” said Tang, the lead author of the paper.

The knot-like shape of the toroid, along with interlocking bumps in the protein envelope, may keep the DNA wedged into the capsid until the virus docks onto the host cell.

“It’s poised in this tube waiting to go through the bacterial wall,” Baker said. “All of the components work together to create an infection machine.”

Source: University of California – San Diego

DNA Poised for Release in Bacteriophage ø29. Jinghua Tang, Norman Olson, Paul J. Jardine, Shelley Grimes, Dwight L. Anderson, and Timothy S. Baker. Structure. June, 2008: 16 (6).

Josh says:

Biology continues to amaze us. It makes sense for the DNA to be tightly packed and under a lot of pressure, thereby ensuring it is effectively delivered to the host upon infection. I’m sure there was a strong selection in favor of the viral particles that did this the best.

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

8 new human genome projects offer large-scale picture of genetic difference

A nationwide consortium led by the University of Washington in Seattle has completed the first sequence-based map of structural variations in the human genome, giving scientists an overall picture of the large-scale differences in DNA between individuals. The project gives researchers a guide for further research into these structural differences, which are believed to play an important role in human health and disease. The results appear in the May 1 issue of the journal Nature.

The project involved sequencing the genomes of eight people from a diverse set of ethnic backgrounds: four individuals of African descent, two of Asian descent, and two of European background. The researchers created what’s called a clone map, taking multiple copies of each of the eight genomes and breaking them into numerous segments of about 40,000 base pairs, which they then fit back together based on the human reference genome. They searched for structural differences that ranged in size from a few thousand to a few million base pairs. Base pairs are one of the basic units of information on the human genome. … Continue Reading »

Enzyme complex could be key to new cancer treatments

Penn State scientists are the first to observe in living cells a key step in the creation of adenine and guanine, two of the four building blocks that comprise DNA. Also called purines, the two building blocks are essential for cell replication. The findings, which will be published in the 4 April 2008 issue of the journal Science, could lead to new cancer treatments that prevent cancer cells from replicating by interfering with their abilities to make purines.

The group used cervical cancer cells–which have an increased demand for purines due to their rapid rates of replication–to demonstrate that a group of six enzymes is involved in the creation of purines. “Our research shows that these enzymes form a cluster prior to purine formation,” said Erin Sheets, an assistant professor of chemistry and a collaborator on the project. … Continue Reading »