Friday, September 14, 2007

1996 Elusive Genetic Switch At Last Yields Image Of Its 3-D Structure1996

March 5, 1996
Elusive Genetic Switch At Last Yields Image Of Its 3-D Structure

IN an arduous feat that involved luck as well as dogged persistence, researchers have worked out the three-dimensional structure of one of the most famous proteins in the history of molecular biology.

The protein, called the lactose repressor, attaches itself to DNA, the genetic material, and physically blocks access to a set of bacterial genes needed to break down milk sugar, or lactose. Only when this protein has been pried loose from the DNA can bacteria make the enzymes they need to digest lactose.

But interest in this protein has little to do with its mundane job of controlling lactose digestion by bacteria. Instead, the protein has taken on a role that is larger than life, becoming the prototype for understanding gene regulation, how genes are turned on and off. As such, it has been studied intensively since the dawn of molecular biology more than 50 years ago.

Yet such is the difficulty of determining three-dimensional molecular structures that biologists had learned virtually everything there is to know about how the protein works but, until now, were still uncertain exactly what it looks like.

Dr. Ponzy Lu of the University of Pennsylvania, who, with Dr. Mitchell Lewis and their colleagues, determined the structure, says the protein looks something like two spring-handled tongs that open when the handle is squeezed, tied together at the handle end with a ribbon. The pincers of the tongs tightly grip two segments of DNA to keep the lactose genes from functioning.

To open the pincers and free the lactose-digestion genes, a sugar molecule inserts itself into the tongs. Like human fingers pressing the tongs, the sugar presses the protein, forcing it to release the DNA.

The newly discovered structure, in glowing computer-generated color, is on the cover of the current issue of the journal Science.

Dr. Lewis said the discovery meant that investigators could start thinking about how to redesign the lactose-repressor protein so it could be used to control other genes selectively.

Dr. Thomas A. Steitz, a professor of molecular biochemistry and biophysics at Yale University, said that the complete structure of the protein validated an approach that he and others had used to get an approximation of the structure of very large and complex molecules that are hard to work with. They have analyzed them piecemeal, in sort of modular form, then put the pieces together. With the lactose-repressor protein, Dr. Steitz said, one of his inferences of the three-dimensional structure, published in Science last year, turned out to be essentially correct.

Dr. Kathleen S. Matthews, a biochemistry and cell biology professor at Rice University who wrote a commentary accompanying the paper by Dr. Lu and Dr. Lewis in Science, added that the fact that researchers who tried to infer the structure from incomplete information came up with close approximations of the protein's structure "bodes well because there are proteins we will not crystallize quickly."

Nonetheless, Dr. Matthews said, there were surprises in the structure. In particular, she said, she was struck by the way the protein grabbed the two pieces of DNA and pushed them together. "The closeness of these two sites is not what anyone would have predicted," she said.

Dr. Sankar Adhya, a molecular biologist at the National Cancer Institute, said that the structure of the lactose-repressor protein should remind scientists that molecules interact in three dimensions. He said that in researchers' enthusiasm to find the DNA sequence of genes, they often forgot that they were getting just a one-dimensional picture of something that functions in three dimensions.

Despite the importance of learning what molecules actually look like in three dimensions, finding the structures of large molecules tends to be a Herculean task.

"A crystallographic laboratory needs to generate structures to renew grants," Dr. Adhya said. So, he added, "difficult problems like the lac repressor tend to be put aside."

The saga of the lactose repressor began in 1942, before DNA was even known to be the genetic material. Two French scientists, Dr. Francois Jacob and Dr. Jacques Monod, were studying sugar metabolism in bacteria and noticed a curious thing. Bacteria, given a choice of sugars, digest them in a rank order, going from the simplest one -- glucose -- and moving on, step by step, toward the more complex sugars.

"That was a puzzle," Dr. Lu said. "You would think that since bacteria have holes in their walls and everything goes in, they would use everything."

Dr. Jacob and Dr. Monod pursued the problem, studying mutations in bacteria that altered their sugar metabolism. Finally, they deduced that there must be a protein, the lactose repressor, that turns off lactose-digestion genes when they are not needed and turns them on when lactose concentrations outside the cell get high. Their paper quickly became a classic, "probably one of the most elegant papers in molecular biology," Dr. Lu said, and it won them the Nobel Prize in 1961.

From there, molecular biologists became obsessed with the lactose repressor. They wanted to know how it worked and what the DNA that it bound to looked like. Their studies elucidated the ways that genes control protein synthesis in cells as well as the way proteins, by binding to DNA, can determine which genes are active. The work led to the first isolation of a gene in the laboratory, a result that so troubled its principal researcher, Dr. James Shapiro of the University of Chicago, that he withdrew from science temporarily, fearful of what molecular biology had wrought.

Dr. Lu provided a list of firsts in molecular biology that came from work on the lactose repressor: The first DNA sequence found was the segment that is bound by the repressor. The first experiment to show that genetic engineering was possible used the lactose system to control the synthesis of a hormone gene that had been added to bacteria. The first gene sequenced was the gene for the lactose repressor.

To understand the lactose repressor and how it functions, Dr. Jeffrey H. Miller, a professor in the department of microbiology and molecular genetics at the University of California at Berkeley, and his colleagues produced more than 4,000 mutations of the protein. Each mutation involved substituting a single building block of the protein with another one, then seeing how that pinpoint change affected the protein's ability to function.

Dr. Miller's ultimate goal, he said, has been to see the three-dimensional structure of the protein and to put that together with what he had learned from his mutations to discover "what is important in the structure and how it works."

Others, too, have wanted to see the structure of the lactose-repressor protein, this protein that, Dr. Lu said, is "central to the epistemology of molecular biology."

To get a detailed picture of a protein, researchers first must make crystals of the protein. Then they bombard the crystals with X-rays, and they record the pattern of spots made by X-rays on film as they bounce off the atoms that make up the protein crystal. From that information, they must use complicated mathematical and computational methods to reconstruct the arrangement of atoms that gave rise to the pattern.

The problem with the lactose-repressor protein, however, was that it was so huge and so wiggly that it seemed impossible to crystallize. For nearly 30 years, researchers had tried in vain to make crystals. At one point, Dr. Lu and his colleagues even tried to grow crystals in the zero gravity of space, twice sending a solution of lactose-repressor protein on space shuttle missions, but to no avail. The crystals that grew were too small to be useful.

As an interim solution, investigators noticed that if they clipped off the wiggling part of the protein -- the part that binds to DNA -- they could get a crystal of what was left behind. In 1978, Dr. Steitz and his colleagues determined the three-dimensional structure of the part that could be crystallized. But Dr. Steitz noted at the time that it was almost like saying he was going to study brain function by studying decapitated rats.

Dr. Lu and his colleagues spent five years trying various chemical tricks to coax crystals to form and to find ways to incorporate reference atoms into the crystals so they could locate the exact position of molecules. Finally, they managed to make the crystals, and they hit upon a nickel compound that they could incorporate into the molecules to help them analyze the data. Their ultimate success, Dr. Lewis said, was really due to "brute force."

The data from the X-ray bombardment of this large molecule were complex and difficult to interpret. Dr. Lewis ended up inventing a new computational method, which he calls a genetic algorithm, that allowed the investigators to find the molecular structure implicit in the mass of data.

Now, Dr. Matthews said, she and others will be marveling at how this protein's three-dimensional form adds to their understanding of how proteins recognize and make contact with DNA -- the fundamental aspect of turning genes on and off. As the structure of the lactose-repressor protein shows, she said, such interactions "may involve bending and protein folding and unfolding that you don't see unless you get up close and personal with the protein."

Correction: March 9, 1996, Saturday

An article in Science Times on Tuesday about research that determined the three-dimensional structure of an important protein, the lactose repressor, misstated an affiliation of Dr. Jeffrey H. Miller, a researcher who has worked on the protein. He is on the faculty at the University of California at Los Angeles, not Berkeley.

* Copyright 2007 The New York Times Company

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