Berkeley Lab Highlights
Bioscience: Genetic Keystone Revealed in 3-D

The TF protein complex will then make contact with and begin to move across the exposed DNA strands. The TFIID protein is the keystone of the complex because it is able to recognize precisely where along the DNA a gene�s message begins. At that point, the TFIID protein will stop and bind the entire TF complex to the gene. It then interacts with another protein called RNA polymerase to transcribe the genetic code into messenger RNA, the vessel by which genetic information is transported out of a cell�s nucleus and into its cytoplasm where proteins are assembled.

The research team that produced the 3-D images and model of the TF complex was led by Eva Nogales, who holds a joint appointment with Berkeley Lab�s Life Sciences Division (LSD) and UC Berkeley�s Molecular and Cell Biology Department. Other members of the team were LSD�s Frank Andel, and Andreas Ladurner, Carla Inouye, and Robert Tjian of UC Berkeley. Their results were published in the December 10, 1999 issue of the journal Science.

Evangelina Nogales de la Morena is a true international scholar. A native of Madrid, she received her undergraduate degree in physics from the Universidad Autonoma de Madrid, and her Ph.D. from the University of Keele in England. She came to Berkeley Lab as a post-doctoral fellow and is now a staff scientist here, and an assistant professor at UC Berkeley with her own laboratory.

Frank Andel III earned his bachelor�s and master�s degrees at the University of Alaska in Fairbanks then came to UC Berkeley for his Ph.D. He was one of the first post-docs to join the Nogales� lab.

Said Nogales, "Our 3-D reconstruction gives us a good idea as to how TFIID works in concert with TFIIA and TFIIB to initiate and regulate the transcription of protein coding genes." 

Structural determinations for some domains and other components of the TFIID, TFIIA and TFIIB proteins had been previously determined through the use of x-ray crystallography. However, the size of TFIID, coupled with the difficulties posed in trying to crystallize all of the TF proteins together had precluded the use of x-ray diffraction for imaging the entire complex. Consequently, until the work of Nogales and her colleagues, the overall shape and relative position of the components within the TF complex had remained a mystery.

Solving a protein�s structure via electron microscopy and single-particle image analysis does not require that the protein be crystallized. Because the Berkeley researchers  did not have to crystallize the TF complex, they could work with a relatively small amount of sample and still produce a 3-D image of the entire transcriptional machine. In their Science paper, they presented TFIID as roughly 200 x 135 x 100 angstroms in size, dominated by three main lobes that are the same size (60 angstroms) but differ in structural detail. The lobes are arranged around a cavity whose open channel, measuring 40 angstroms, can easily fasten around a single strand of DNA.

"The electron microscopy studies presented here provide a model for the structure of the TFIID, TFIIA and TFIIB complex in the absence of DNA," said Nogales. "Antibody mapping of the TATA Binding Protein (TBP) within TFIID strongly suggests the binding position of DNA to be at the top of the central cavity within the TFIID complex."

To produce the 3-D model, team-member Andel recorded thousands of images of randomly-oriented individual protein molecules in purified samples obtained from the separate research group that is led by Tjian. A computer was then used to align these thousands of randomly-oriented images into an ordered array and merge them into a three-dimensional reconstruction. In essence, the single-particle image analysis was used to create a virtual crystal.

Said Nogales, "Our work showed that electron microscopy is a good technique for studying biological complexes of proteins and nucleic acids that are too large or too fragile to be crystallized for x-ray diffraction studies. It also showed that single-particle image analysis is a useful technique for the structural characterization of mega-dalton transcription complexes."

At a resolution of 35 angstroms the shapes of TFIID and its companion proteins plus their relative positions within the TF complex could clearly be seen. When this electron microscopy information is combined with the x-ray data on various substructures within the complex�a technique dubbed "hybrid crystallography"�Nogales and her colleagues expect to find further clues as to how the transcriptional machinery comes together.

"Our goal is to characterize the structure of the functional TFIID complex and other large transcriptional cofactors in order to gain crucial information on the interaction between its different components," Nogales said. "This is required to understand the mechanism of gene transcription regulation."

Nogales is part of another team that is exploring the viability of applying the enormous computational powers of a supercomputer to electron microscopy imaging techniques and single-particle image analysis. With a supercomputer, rather than working with a few tens of thousand images as is now done, it should be possible to collect and merge as many as a million images of a single non-crystallized protein, then reconstruct these images into a 3-D model at a resolution comparable to that achieved with x-ray crystallography. What�s more, with supercomputers such as those at Berkeley Lab�s National Energy Research Scientific Computing Center (NERSC) which are capable of making trillions of calculations every second, such reconstructions could be done in less than half a day.

Robert Glaeser, a biophysicist with Berkeley Lab�s Physical Biosciences Division who is a pioneer and world authority on electron crystallography and one of the three collaborators with Nogales on this project�the others are LSD biophysicist Kenneth Downing and Ravi Malladi and Esmond Ng of NERSC�says the idea is the equivalent of using a computer to perform the difficult task of protein crystallization. He calls this approach "crystallization in silico." � Lynn Yarris

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