NEWS RELEASE, 12/09/99

UC Berkeley, LBNL scientists snap first 3-D pictures of the "heart" of the transcription machine

By Robert Sanders, Public Affairs

BERKELEY-- Scientists at the University of California, Berkeley, and Lawrence Berkeley National Laboratory (LBNL) have obtained the first good picture of a major chunk of the machinery that turns genes on and off.

With the help of electron microscopy and a relatively new technique called single particle image analysis, the researchers reconstructed a three-dimensional picture of the heart of the machine - the part that binds to DNA and starts the process of gene transcription.

The picture shows for the first time how the proteins are arranged, and gives clues to the inner workings of the machinery that transcribes genes - the complex of proteins that latches onto and copies DNA into an RNA blueprint for building proteins.

"For now, this is very basic, very fundamental research that helps us understand how the machine works," said team leader Eva Nogales, an assistant professor of molecular and cell biology at UC Berkeley and a scientist at LBNL. "But it has implications for the treatment of disease, since essentially treatment comes down to modifying the behavior of proteins. One way to do that is to regulate transcription, which is why proteins involved in transcription are a major target for drug development."

The results will appear in the Dec. 10 issue of the journal Science.

The entire machine that transcribes a gene is composed of perhaps 50 proteins, including RNA polymerase, the enzyme that converts DNA code into RNA code. A crew of transcription factors grabs hold of the DNA just above the gene at a site called the core promoter, while associated activators bind to enhancer regions farther upstream of the gene to rev up transcription.

Working as a tightly knit machine, these proteins transcribe a single gene into messenger RNA. The messenger RNA wends its way out of the nucleus to the factories that produce proteins, where it serves as a blueprint for production of a specific protein.

The new detail is of the proteins forming the very large complex that binds DNA.

"We've never had a picture of this entire complex, and it tells us a lot about how this huge molecular machine works," said Robert Tjian, professor of molecular and cell biology and an author of the paper. The vast majority of work reconstructing the cell's transcription machinery has been done by Tjian and his colleagues at UC Berkeley over the past two decades. Both Tjian and Nogales are part of UC Berkeley's Health Sciences Initiative, a research effort that draws scientists from both the physical and biological sciences into the search for solutions to today's major health problems.

Tjian and Nogales admit that the picture now revealed is the first step in a long-term project to determine the three-dimensional arrangement of all the proteins in the machine, in enough detail to see the individual amino acids that make up each protein.

"The resolution we have now is good enough for learning about how things happen in the cell, but drug design comes with atomic modeling at a much finer resolution - about 10 times better than we have now," Nogales said.

Nogales now is at work sharpening this picture of the complex. She and Andel could not use standard X-ray crystallography to determine the structure of these proteins because the complex is about 10 times too big and the quantities they can obtain from the nucleus about a thousand times too small for that technique.

Transcription begins with the binding of a subunit called transcription factor IID, or TFIID, to DNA in the nucleus. TFIID contains a protein that homes in on a sequence of nucleic acids denoted TATA, which is found in the core promoter regions of all genes in higher organisms, or eukaryotes. The protein that binds to the TATA sequence has been dubbed the TATA binding protein.

Tjian's lab, which discovered the components of TFIID some 10 years ago, purified enough of it to give to Nogales, a biophysicist and structural biologist. Nogales's postdoctoral fellow Frank Andel III took a series of electron microscope pictures of the large complex, and from the random orientations in the pictures was able to reconstruct its three-dimensional shape.

Subsequently, they added to the complex another of the proteins, dubbed TFIIB, and used the same image analysis technique to determine how it binds to the larger TFIID complex. They then added a third, dubbed TFIIA, to determine where it binds. Finally they pinpointed the location of the TATA binding region using antibodies to the binding protein.

The resulting picture is of a three-lobed structure, not unlike what they expected.

"The complex looks like a horseshoe or a crab claw," Nogales said. "It is flexible and can go around the DNA and grab on."

"It is reassuring to see that the relative positions of transcription factors IIA and IIB are where we thought they would be," Tjian said.

The image analysis technique used by Nogales' team provides a fairly low resolution image, showing detail no smaller than about 35 Angstroms across. (An atom is about one Angstrom across.) However, using state-of-the-art cryoelectron microscopy, she hopes to get down to atomic scales.

"With cryoelectron microscopy, we now can get down to a resolution of 7 Angstroms, but because the technique is evolving very, very fast, I hope we can soon get down to detail of 3 Angstroms or better," Nogales said.

As Nogales and Andel work toward better resolution, the Nogales and Tjian labs will continue to add more proteins to the complex to reconstruct the 3-D structure of the whole transcription machine.

"We plan to build it up one molecule at a time," she said.

Coauthors with Nogales, Tjian and Andel are postdoctoral fellows Andreas G. Ladurner and Carla Inouye. Both work with Tjian.

The work was supported in large part by a Laboratory Director's Research and Development Grant from LBNL and the Department of Energy. Nogales also has support from the National Institute of General Medical Sciences of the National Institutes of Health.


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