Genes may contain the blueprint for life, but they’re written more like coded messages in a spy thriller: Short segments of DNA carrying instructions for protein formation are imbedded in a longer text, and interspersed with “filler” DNA that has no known function. The picture becomes still more complicated when one realizes that the useful “characters” in the genetic code may be pieced together, or spliced, in different combinations. “Alternative splicing” allows relatively few genes to give rise to a great number of protein structures.

Since the discovery of RNA splicing, around 25 years ago, scientists have worked to understand how the right sequences are lifted out and strung together to make a coherent set of instructions. Both straightforward and alternative splicing take place in the “spliceosome,” situated in the cell nucleus. A large complex of proteins and short strands of RNA, the spliceosome distinguishes the beginnings and ends of coded segments, precisely cutting and “stitching” them together.
 

A husband-and-wife team, Prof. Ruth Sperling of the Hebrew University of Jerusalem’s Genetics Department and Prof. Joseph Sperling of the Weizmann Institute’s Organic Chemistry Department, has produced the most detailed 3-D representation of the spliceosome’s structure to date. Rather than follow others’ attempts to observe spliceosomes created in test tubes, the Sperlings and team members Maia Azubel, Ruth’s graduate student, and Sharon Wolf of the Institute’s Chemical Research Support Department managed to take spliceosomes directly from living cells and examine them under an electron microscope. 
 

The living spliceosome presented them with a challenge. In cells they come packaged in sets of four identical modules strung together like beads on a strand of RNA, each a miniature spliceosome capable of splicing on its own. The connections between the modules tend to be flexible, allowing the position of the units to vary in relation to one another, and making pinning down a definitive shape and structure for the whole complex close to impossible.

The team found a way to cut the RNA connections between the modules without harming the integral short strands of RNA that are essential to the splicing process, so they could study them individually. Split-second freezing at very low temperatures allowed the scientists to view the spliceosome units in a state as close to natural as possible. From thousands of images, each at a slightly different angle, a composite 3-D structure of the spliceosome was built up. 

The revealed structure has two distinct, unequal halves surrounding a tunnel. The larger part appears to contain proteins and the short segments of RNA, while the smaller half is made up solely of proteins. On one side the tunnel opens up into a cavity, which the researchers think functions as a holding space for fragile RNA waiting to be processed in the tunnel itself.

What they didn’t see may be as important as what they saw. Whereas researchers examining splicing in test tubes saw evidence of a complicated sequence of events in which the spliceosome machinery assembles itself anew for each splicing job, the team’s investigations of spliceosomes from live cells found splicing to take place in preformed machines. This fits in with what is known about the way cells optimize their workload.  “It’s much more efficient to have a machine on hand, ready to go, than to build a new one each time,” they noted.

Prof. Joseph Sperling’s research is supported by the Helen and Milton A. Kimmelman Center for Biomolecular Structure and Assembly; the J & R Center for Scientific Research; the Joseph and Ceil Mazer Center for Structural Biology; and Lois Zoller, Chicago, IL. Prof. Sperling is the incumbent of the Hilda Pomeraniec Memorial Chair of Organic Chemistry.

"Jack of all trades, master of an unknown,” is how Dr. Dan Tawfik of the Biological Chemistry Department describes a multi-purpose enzyme, nicknamed PON. The enzyme performs a variety of jobs in the body, including breaking down harmful chemicals found in pesticides and nerve gases and ridding the arteries of plaque-forming clumps of LDL (“bad cholesterol”) that can lead to arteriosclerosis. But these are just sidelines for PON, whose original function remains shrouded in its evolutionary past. Not only does it “moonlight,” accomplishing various jobs on the side, but it performs them inconsistently in different people, doing a sloppy job for some, a more efficient one for others. This is due to variations in its encoding gene. Referred to as polymorphism, this multiplicity of forms means, for instance, that some people’s PONs are up to 50 times more active than those of others.

How does one enzyme display such diverse performances? Very little was known about its workings when Tawfik and postdoctoral fellow Dr. Amir Aharoni decided to take a closer look.

To examine protein function, scientists apply crystallization, a process that yields X-ray images that are then translated into 3-D models. But crystallizing PON enzymes filtered from the blood - a process that yields only minuscule amounts - had proven difficult. So the team set out to create pure, genetically engineered proteins, generated in large enough quantities to crystallize and analyze them in detail.

The method they used, known as “directed evolution,” works on two principles of natural, Darwinian evolution: genetic diversity and survival of the fittest. Genetic diversity was created in the lab by inducing random mutations in the gene encoding the PON enzyme. The mutated enzymes were then inserted into bacterial cells, which were allowed to grow and multiply. Selection took place in the lab, with the scientists substituting for nature, deciding which were “fit” according to certain criteria, recombining them with other mutations and selecting again. Eventually, they found several versions of the enzyme that proved ideal for undergoing the procedures needed to solve its structure.

Studying the bacterial forms of the protein, the team succeeded in revealing many aspects of its function, including how its various forms in people differ in their ability to perform assigned tasks. They discovered, for instance, that the enzyme is shaped something like a six-bladed propeller and that modifications in its structure tend to take place near its active site (which performs the actual work of the enzyme) in a way that causes instability in the scaffold of the enzyme. They also got a good glimpse of how PON “roosts” on the HDL (“good cholesterol”) and the role it may play in sucking up oxidized lipids from the LDL (“bad cholesterol”) in arteries.

Once the main structure was solved, the research team tackled a new goal: that of creating new PON variants that would be even better at specific tasks than naturally occurring ones. Again using directed evolution, they came up with enzymes that could specialize in the chemical clean-up of harmful pesticides or reduce potential risk factors in heart disease. Interestingly, as the enzyme specialized in one task, it lost its ability to do others. “It’s similar to the jack-of-all-trades who takes up carpentry. Eventually he forgets how to do the plumbing and tile-laying,” says Tawfik.

Now that the team has a better picture of how differences in PON’s structure affect its actions in the test tube, they plan to focus on how it works inside the body. Research in this field might advance the treatment of arterio-sclerosis as well as of neuronal damage arising from exposure to harmful chemicals.

Other scientists collaborating in this research are Leonid Gaidukov of the Biological Chemistry Department; Prof. Israel Silman and Lilly Toker of the Neurobiology Department and Prof. Joel Sussman and Dr. Michal Harel of the Structural Biology Department.

Form fits function

To the uninitiated, diagrams of protein structures may look like colorful tangles of ribbon or free-style children’s art projects. Yet in proteins, as in architecture, form follows function.

Each protein consists of amino acids linked together in a long chain that folds and twists into the highly intricate shape in which it performs its specialized function.

When Sir John Kendrew and Max Perutz were awarded the Nobel Prize in 1962 for solving the first three-dimensional structures of proteins, they had spent over twenty years on the task. Even with today’s improved methods, much trial-and-error work is still involved, and scientists may spend months, even years, solving a protein’s structure.

The Israel Structural Pro-teomics Center (ISPC), based at the Weizmann Institute and headed by Prof. Joel Sussman of the Structural Biology Depart-ment, has made streamlining this process its main goal. Having tackled up to 30 proteins at a time in its pilot year, the Center intends to raise that number to several hundred in the coming years. Supported by the Ministry of Science, Culture and Sports, the ISPC facility is open to scientists throughout Israeli academia and industry interested in solving protein structures.

A major partner in a network of European structural proteomics centers (which Weizmann scientists were influential in establishing), the ISPC aspires to lead the way in protein research and open up new avenues in drug design and disease treatments.

Dr. Tawfik’s research is supported by the Y. Leon Benoziyo Institute for Molecular Medicine; the Dolfi and Lola Ebner Center for Biomedical Research; the Estelle Funk Foundation; the Dr. Ernst Nathan Fund for Biomedical Research; the Henry S. and Anne Reich Family Foundation; the Harry and Jeanette Weinberg Fund for Molecular Genetics of Cancer and the Eugene and Delores Zemsky Charitable Foundation, Inc. He is the incumbent of the Elaine Blond Career Development Chair.