Hello to all;
 
One more salvo from the news-hound. I think this one could be the
start of something big.
 
Ain't modern technology grand?
 
Janet
the Parkie in Paradise
where the loquat trees are heavy with fruit.
 
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Elusive genetic switch finally yields image of 3-D structure
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Copyright ) 1996 Nando.net   Copyright ) 1996 N.Y. Times News Service
 
(Mar 5, 1996 02:58 a.m. EST) 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 about 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 snail, or barbecue, tongs 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 prying
apart the tongs, the sugar opens 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.
 
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, Steitz said,
his inference 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 Lu
and Lewis in Science, added that the fact that researchers who tried
to infer the structure from incomplete information were for the most
part accurate in their guesses about the protein's structure "bodes
well because there are proteins we will not crystallize quickly."
 
Nonetheless, 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," 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," Lu said. "You would think that since bacteria
have holes in their walls and everything goes in, they would use
everything."
 
Jacob and 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," 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.
 
Lu provided a list of firsts in molecular biology that came from work
on the lactose repressor: The first DNA sequence that was 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 that was 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.
 
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, 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, 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, Steitz and his colleagues determined the three-dimensional
structure of the part that could be crystallized. But Steitz noted at
the time that it was almost like saying he was going to study brain
function by studying decapitated rats.
 
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, 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. 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, Matthews said, she and others will be marveling at how this
protein's actual 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."
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-----Janet [log in to unmask]