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RNA inner workings partly unveiled in Stanford study

13.02.2004


In the world of molecules, DNA tends to get top billing at the expense of RNA, which is critical for turning DNA’s genetic blueprint into working proteins. Researchers at the Stanford University School of Medicine have published significant insights into how the RNA molecule completes this task in two back-to-back papers in the Feb. 13 issue of Science.



All the genetic information contained in DNA is silent, said Roger Kornberg, PhD, the Mrs. George A. Wizner Professor in Medicine and professor of structural biology. What gives it a voice is RNA polymerase, the enzyme that copies DNA into RNA through a process called transcription. Along with more than a dozen helper molecules, RNA polymerase determines which proteins are produced within a cell. But before scientists can understand the transcription process, they must first unveil the inner structure of RNA polymerase.

Kornberg’s lab has been studying RNA and the enzyme that makes it for more than 20 years. Past studies from the lab have shown that the machinery of the RNA polymerase system is in three layers. Kornberg’s group published groundbreaking findings in 2001 outlining the structure of the innermost layer. The two current papers focus on the middle layer, which contains many of the helper molecules.


To see the structure of the protein layers, the group passed extremely bright X-rays - generated at the Stanford Synchrotron Radiation Laboratory, or SSRL - through a crystallized version of the proteins. The crystal scatters the X-rays, generating a distinctive diffraction pattern that reveals the sample’s three-dimensional atomic structure.

Part of their current work looked at RNA polymerase along with one of the five helper molecules, called transcription factors, in the middle layer. From the structure that could be seen when just a single transcription factor was added, the team extrapolated a picture of the entire middle layer, which, Kornberg said, enabled them to understand how the enzyme locates a gene along a stretch of DNA.

At the level of detail the group obtained, some intriguing structures came to light, offering the first real understanding of the defining events of transcription. They saw a docking site that might reveal the starting point of transcription, a spot where the RNA polymerase is correctly situated on a gene. They also saw something completely unexpected: a "finger" of the helper factor protein that pokes into the enzyme’s active center. The researchers speculate that the poking action may help slow down the transcription process so that the strands of DNA and newly made RNA can separate properly.

"This turned out to be quite interesting. No one had even speculated about it before," said David Bushnell, PhD, a research associate and first author of one of the papers. "We think the protrusion reaching into the enzyme makes sense of a lot of genetic and biochemical data that people were scratching their heads over. Figuring out the structure gave remarkable context to years of hard work by many people."

The second paper describes how the team caught a snapshot of the polymerase in action, something that hadn’t been done before. Kenneth Westover, an MD/PhD student and first author of the second paper, developed a method in which the newly made RNA could be visualized coming off the DNA.

"When we look to see where the two separate, we find that lo and behold, the RNA passes through a hole and the DNA comes out over the top," said Kornberg. "The separation that is achieved at the hole is revealed for the first time in this paper."

How the strands of RNA and DNA are pushed apart has a simple physical explanation: the RNA polymerase inserts itself as a wedge between the two, with the RNA trailing out the hole. That same opening is the one that the protein finger dips into. "One might have imagined this, but to see it is another thing entirely," said Kornberg.

"These two papers are both quite astonishing in what they reveal," he added. "One because it shows us this protein finger that pokes through and because we can intuit all the rest of the structure around the polymerase, and the other paper because it shows this amazing dynamic mechanism by which the RNA is separated from DNA."

To find good diffracting crystals out of the hundreds made, the researchers used a new automatic robotic screening system developed at SSRL with grants from the National Institutes of Health. The automated screening system stores the tiny frozen crystals on nylon loops at the end of metal pins. A robotic arm retrieves each pin and aligns the crystal in the path of the X-ray beam. The robot can automatically test 300 samples without the need for researchers to carry out a manual transfer for each sample as was done in the past.

"It saves a lot of time while optimizing the quality of the data," said SSRL scientist Mike Soltis, PhD, head of the macromolecular crystallography group. "With the new system, the Kornberg group screened 130 crystals in seven hours without losing any. Two weeks earlier, they had manually mounted 100 crystals in 24 hours, losing a few crystals and much sleep in the process."

The Kornberg group plans to build upon their findings and continue to explore the inner workings of RNA polymerase. "Because we have overcome technical problems in making the complexes, it opens a huge opportunity for a lot of other variations of this. We can do a lot of experiments that we couldn’t do before," said Westover.


Stanford University Medical Center integrates research, medical education and patient care at its three institutions - Stanford University School of Medicine, Stanford Hospital & Clinics and Lucile Packard Children’s Hospital at Stanford. For more information, please visit the Web site of the medical center’s Office of Communication & Public Affairs at http://mednews.stanford.edu.

PRINT MEDIA CONTACTS:
Mitzi Baker at 650-725-2106 (mitzibaker@stanford.edu)
Amy Adams at 650-723-3900 (amyadams@stanford.edu)

Mitzi Baker | EurekAlert!
Further information:
http://med-www.stanford.edu/MedCenter/MedSchool/
http://mednews.stanford.edu

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