Posts Tagged ‘molecular biology’

Scientists capture most detailed images yet of tiny cellular machines

 :: Posted by American Biotechnologist on 06-03-2014

A grandfather clock is, on its surface, a simple yet elegant machine. Tall and stately, its job is to steadily tick away the time. But a look inside reveals a much more intricate dance of parts, from precisely-fitted gears to cable-embraced pulleys and bobbing levers.

Like exploring the inner workings of a clock, a team of University of Wisconsin-Madison researchers is digging into the inner workings of the tiny cellular machines called spliceosomes, which help make all of the proteins our bodies need to function. In a recent study published in the journal Nature Structural and Molecular Biology, UW-Madison’s David Brow, Samuel Butcher and colleagues have captured images of this machine, revealing details never seen before.

In their study, they reveal parts of the spliceosome — built from RNA and protein — at a greater resolution than has ever been achieved, gaining valuable insight into how the complex works and also how old its parts may be.

By better understanding the normal processes that make our cells tick, this information could some day act as a blueprint for when things go wrong. Cells are the basic units of all the tissues in our bodies, from our hearts to our brains to our skin and lungs.

It may also help other scientists studying similar cellular machinery and, moreover, it provides a glimpse back in evolutionary time, showing a closer link between proteins and RNA, DNA’s older cousin, than was once believed.

“It gives us a much better idea of how RNA and proteins interact than ever before,” says Brow, a UW-Madison professor of biomolecular chemistry.

The spliceosome is composed of six complexes that work together to edit the raw messages that come from genes, cutting out (hence, splicing) unneeded parts of the message. Ultimately, these messages are translated into proteins, which do the work of cells. The team created crystals of a part of the spliceosome called U6, made of RNA and two proteins, including one called Prp24.

Crystals are packed forms of a structure that allow scientists to capture three-dimensional images of the atoms and molecules within it. The crystals were so complete, and the resolution of the images so high, the scientists were able to see crucial details that otherwise would have been missed.

The team found that in U6, the Prp24 protein and RNA — like two partners holding hands — are intimately linked together in a type of molecular symbiosis. The structure yields clues about the relationship and the relative ages of RNA and proteins, once thought to be much wider apart on an evolutionary time scale.

“What’s so cool is the degree of co-evolution of RNA and protein,” Brow says. “It’s obvious RNA and protein had to be pretty close friends already to evolve like this.”

The images revealed that a part of Prp24 dives through a small loop in the U6 RNA, a finding that represents a major milestone on Brow and Butcher’s quest to determine how U6′s protein and RNA work together. It also confirms other findings Brow has made over the last two decades.

“No one has ever seen that before and the only way it can happen is for the RNA to open up, allow the protein to pass through, and then close again,” says Butcher, a UW-Madison professor of biochemistry.

Ultimately, Butcher says they want to understand what the entire spliceosome looks like, how the machines get built in cells and how they work.

While this is the first protein-RNA link like this seen, Brow doesn’t believe it is unique. Once more complete, high-resolution images are captured of other RNA-protein machines and their components, he thinks these connections will appear more commonly.

He hopes the findings mark a transition in the journey to understand these cellular workhorses.

“It’s exciting studying these machines,” he says. “There are only three big RNA machines. Ours evolved 2 billion years ago. But once it’s figured out, it’s done.”

The U6 crystal structure was imaged using the U.S. Department of Energy Office of Science’s Advanced Photon Source at Argonne National Laboratory. The work was funded by a joint grant from the National Institutes of Health shared by Brow and Butcher.

Thanks to University of Wisconsin-Madison for contributing this story.

Suprise! mRNA and Protein Levels Do Not Always Correspond!

 :: Posted by American Biotechnologist on 03-27-2014

The central dogma of molecular biology states that DNA codes for RNA and RNA codes for protein. It was widely understood that because protein is translated from mRNA, the amount of mRNA in a cell would somewhat correspond to the quantity of cellular protein. In a new study out of Notre Dame, scientists have shown that this theory is not always correct. While in many cases mRNA and protein levels do correspond, there are a surprisingly high number of exceptions, demonstrating that the amounts of a particular protein can be controlled by multiple mechanisms.

Bioanalytical chemist Norman Dovichi and molecular biologist Paul Huber identified and measured the levels of about 4,000 proteins, which exhibited patterns of expression that reflect key events during early Xenopus development resulting in the largest data set on developmental proteomics for any organism.

The study was conducted in Xenopus laevis embryos, which is a favored model for this type of research. In Xenopus, development takes place in well-defined stages outside the mother, thereby allowing embryogenesis to be monitored in real time. Additionally, embryos develop rapidly, achieving a nearly fully developed nervous system within four days.

Their results are available open access in Scientific Reports.

Powerful New Tool for Studying DNA Elements that Regulate Genes

 :: Posted by American Biotechnologist on 03-24-2014

An international team led by researchers with the Lawrence Berkeley National Laboratory (Berkeley Lab) has developed a new technique for identifying gene enhancers – sequences of DNA that act to amplify the expression of a specific gene – in the genomes of humans and other mammals. Called SIF-seq, for site-specific integration fluorescence-activated cell sorting followed by sequencing, this new technique complements existing genomic tools, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), and offers some additional benefits.

“While ChIP-seq is very powerful in that it can query an entire genome for characteristics associated with enhancer activity in a single experiment, it can fail to identify some enhancers and identify some sites as being enhancers when they really aren’t,” says Diane Dickel, a geneticist with Berkeley Lab’s Genomics Division and member of the SIF-seq development team. “SIF-seq is currently capable of testing only hundreds to a few thousand sites for enhancer activity in a single experiment, but can determine enhancer activity more accurately than ChIP-seq and is therefore a very good validation assay for assessing ChIP-seq results.”

Dickel is the lead author of a paper in Nature Methods describing this new technique. The paper is titled “Function-based identification of mammalian enhancers using site-specific integration.”

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Going Beyond the Central Dogma of Molecular Biology

 :: Posted by American Biotechnologist on 03-18-2014

Our genome, we are taught, operates by sending instructions for the manufacture of proteins from DNA in the nucleus of the cell to the protein-synthesizing machinery in the cytoplasm. These instructions are conveyed by a type of molecule called messenger RNA (mRNA).

Francis Crick , co-discoverer of the structure of the DNA molecule, called the one-way flow of information from DNA to mRNA to protein the “central dogma of molecular biology.”

Yehuda Ben-Shahar and his team at Washington University in St. Louis have discovered that some mRNAs have a side job unrelated to making the protein they encode. They act as regulatory molecules as well, preventing other genes from making protein by marking their mRNA molecules for destruction.

“Our findings show that mRNAS, which are typically thought to act solely as the template for protein translation, can also serve as regulatory RNAs, independent of their protein-coding capacity,” Ben-Shahar said. “They’re not just messengers but also actors in their own right.” The finding was published in the March 18 issue of the new open-access journal eLife.

Although Ben-Shahar’s team, which included neuroscience graduate student Xingguo Zheng and collaborators Aaron DiAntonio and his graduate student Vera Valakh, was studying heat stress in fruit flies when they made this discovery, he suspects this regulatory mechanism is more general than that.

Many other mRNAs, including ones important to human health, will be found to be regulating the levels of proteins other than the ones they encode. Understanding mRNA regulation may provide new purchase on health problems that haven’t yielded to approaches based on Crick’s central dogma.

Read the full story A novel mechanism for fast regulation of gene expression.

Lab on a chip….on a cell phone!

 :: Posted by American Biotechnologist on 11-18-2013

In developing nations, rural areas, and even one’s own home, limited access to expensive equipment and trained medical professionals can impede the diagnosis and treatment of disease. Many qualitative tests that provide a simple “yes” or “no” answer (like an at-home pregnancy test) have been optimized for use in these resource-limited settings. But few quantitative tests—those able to measure the precise concentration of biomolecules, not just their presence or absence—can be done outside of a laboratory or clinical setting. By leveraging their discovery of the robustness of “digital,” or single-molecule quantitative assays, researchers at the California Institute of Technology (Caltech) have demonstrated a method for using a lab-on-a-chip device and a cell phone to determine a concentration of molecules, such as HIV RNA molecules, in a sample. This digital approach can consistently provide accurate quantitative information despite changes in timing, temperature, and lighting conditions, a capability not previously possible using traditional measurements.

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