Picking Up the Pieces

By Jason Socrates Bardi

"Presently I am going to press the lever, and off the machine will go... Have a good look at the thing."

—H.G. Wells, The Time Machine, 1895.

Professor Jamie Williamson, who is a member of the Department of Molecular Biology and The Skaggs Institute for Chemical Biology at The Scripps Research Institute, remembers one day six years ago as if it were yesterday—the day that the ribosome stopped being just a big purple blob.

A big purple blob was how anyone who had studied molecular biology from textbooks published in the mid-1990s knew the shape of the ribosome, the molecular "engine" that drives protein synthesis and is fundamental to the life of all cells in nature. That crude shape was based on electron micrograph images from the 1970s.

But, at the 1998 the triennial ribosome conference meeting in Denmark, several groups presented fairly high-resolution ribosomal structures. On that day, the larger scientific world got its first close-up look at the ribosome.

"It was totally clear that this was a major advance in structural biology," says Williamson. "It's a rare moment in your career when you are right in the middle of something that you know is going to [eventually] win the Nobel Prize."

Not everything seemed so joyous at the time, though.

"It was a time of horror and wonder," admits Williamson, who had been working on the structures of small pieces of the ribosome, aiming to piece them together into a complete picture of the molecule once he was done. "I had the dubious honor of reviewing all the papers, which meant I was qualified to review them but was not publishing any of them. But after awhile, I sobered up and realized that [the new structures] opened up many doors for new kinds of experiments."

It has been a wild ride ever since.

Two Easy Pieces

High-resolution structures of molecules like the ribosome are as important to biologists as detailed blueprints are to mechanics. And just as an auto mechanic needs to have an accurate picture of a car engine to understand how it drives the car, so do biologists need detailed blueprints of the ribosome to understand how it drives protein synthesis.

One of the ancient structures in biology, the ribosome is a huge molecule composed of two subunits, one slightly larger than the other. These are called the 50S and 30S subunits in bacterial ribosomes and the 60S and 40S subunits in mammalian ribosomes—names that date back decades to early biochemical experiments. (The "S" stands for Svedberg, a measure of the sedimentation of a molecule in a centrifuge).

Williamson has been interested in RNA–protein interactions since the early years in his career, when he solved RNA–protein complexes with his laboratory team using nuclear magnetic resonance (NMR). Because of this, the ribosome—which is one big mess of protein and RNA interactions—intrigued him.

The ribosome molecule is a complex of about two-thirds RNA and one-third protein by mass. In bacteria, the large 50S subunit is an amalgam of two RNA chains and 31 different proteins. For the most part, these ribosomes are RNA machines. At their core is the RNA, which does all the important work, holding the two subunits together and recognizing the mRNA, for instance. The proteins are, for the most part, on the outside and contribute to the structural stability of the ribosome as well as recruiting other molecules that are needed for translation.

The 50S subunit is often referred to as the catalytic subunit because it contains the peptidyl transferase center—the part of the molecule that does the chemistry of joining two amino acids together in a growing peptide chain.

The smaller 30S subunit has a single RNA chain and 21 different proteins. Its job is to read the genetic code in messenger RNA (mRNA) and pair the nucleotide codons with their corresponding amino acids.

Studying Ribosome Assembly One Piece at a Time

To study the structure, Williamson and his laboratory divided up the 30S subunit of the ribosome piece-by-piece and sought to solve each piece at high resolution and then stitch the structures of all the pieces together into the low-resolution electron microscope image. Now Williamson is asking different questions, such as how the ribosome is assembled.

In fact, in the few years since the ribosome morphed from a purple blob to a detailed atomic structure, the field has only become more exciting as many scientists have been posing more interesting questions.

Scientists are also further refining their ribosomal structures to get clearer and clearer pictures of it. They can do things not possible a few years ago, such as make crystals of the ribosome, soak it in drugs known to bind to it, and find where these drugs bind. And scientists are solving the structures of ribosomes from mammals and other eukaryotic organisms to compare these to the bacterial ribosomes.

Now that there are structures available for the ribosome, says Williamson, scientists are starting to look at how the structure of the molecule contributes to its function and are reinterpreting decades of biological experiments using the structure. Many questions are still unanswered. It is not exactly clear how the 50S subunit catalyzes peptide bond formation, for instance, nor how the ribosome slides down the mRNA as it translates.

"In the end," he says, "we are doing better experiments now than we ever did before."

Part of his laboratory focuses on the assembly process of the 30S subunit, asking questions focusing on the initial events that cause its 21 proteins to come together with its 1541-base RNA chain, the order of the pieces coming together, and the conformation changes occurring in the process.

He is able to do this because the strategy that he and his colleagues used to make the small pieces of the ribosome for their structural studies was based on an assembly "map" of the ribosome that was created in biochemical studies in the 1960s an 1970s. The scientists who did these studies took the ribosome apart, purifying all its pieces separately and then reconstituting them one-by-one, tinkering with the order in which they mixed the components back together. Because the ribosome self-assembles, by tinkering with this order the scientists were able to discover the steps in which the ribosome assembles.

"What we did [based on this map]," says Williamson, "was to build up a whole series of ever-bigger particles that corresponded to the way in which this thing assembles."

After he saw no need to continue with his structural studies, Williamson realized that he could use the pieces he was working on for folding studies—looking at how pieces twist into the final active conformation and providing a mechanistic framework of how it gets there.

To do this, members of the Williamson laboratory use all the tools of biochemistry, including fluorescence, calorimetry, and mass spectrometry, to observe when the RNA of the 30S subunit folds and when the proteins that are part of it hop on.

"What we're trying to think about is how we can make a conceptual film of the process, and we're trying to understand the fundamental principles that embody what happens," says Williamson. "We can all see the engine under the hood, and now we're trying to understand how it is assembled."

New Targets in HIV

Another part of Williamson's laboratory focuses on the design of new drugs to combat human immunodeficiency virus (HIV).

HIV, the virus that causes AIDS, has a small genome of around 10,000 nucleotides, so it only codes for a handful of proteins. Some of these have familiar-sounding names, like the protease or the reverse transcriptase, because there are already drugs on the market that target them.

Some of the other proteins HIV encodes, like the regulatory proteins Tat and Rev, have not had drugs designed to block them, despite the fact that they are important in the viral lifecycle. Rev, for instance, is a nuclear transport factor that binds to HIV mRNA and is required for getting the full-length HIV RNA out of the nucleus so that it can be translated into the long envelope and capsid proteins and packaged into new virions.

Williamson and his laboratory solved the first structure of Rev bound to RNA in 1996.

Now in a new grant titled "Structural Biology for AIDS-Related Targets" that was funded by the National Institutes of Health (NIH) beginning on October 1 of last year, he and several colleagues at Scripps Research are looking for new or underexploited targets against which to develop novel drugs. In particular, they are attempting to learn how to target the HIV RNA.

"Nobody has really done drug design or drug discovery against an RNA target," says Williamson.

One of the targets that Williamson is looking at is a section of the viral RNA to which the HIV protein Rev binds called the Rev response element (RRE), RRE is a 350 nucleotide section of the viral mRNA transcript that folds up into a stable binding site for Rev. Binding of Rev to RRE is a key stage in viral replication. Significantly, this region seems to be completely conserved—unlike the rest of the HIV genome, it is not prone to mutate.

"In principle," says Williamson, "if you could block this interaction, you could inhibit viral replication."

At the moment, Williamson is collaborating with several other investigators at Scripps Research funded through this grant. These include Assistant Professor Mirko Hennig, who is working with Williamson to set up NMR screens to assay the ability of small molecules to block the interaction between Rev and RRE; Associate Professor Davis Millar, who has a fluorescent screen that can also assay the binding of Rev to RRE; Professor Larry Gerace, who has assays set up in cells to look for Rev function; and Associate Professor Hartmuth Kolb, a chemist who has his own library of compounds and the ability to make analogues of any promising leads they find.

Williamson notes, "All the stuff we're doing [targeting HIV RNA] can be applied to the ribosome to develop new antibiotics."

Full Circle

Antibiotics are the notable exception to the rule that most drugs target proteins. Drugs like tertracycline, kanamycin, and erythromycin all work by targeting bacterial ribosomes. They bind to different parts of the ribosome and interfere with its ability to translate mRNA into protein.

It's not clear how all of these antibiotics do this, but some evidence suggests that certain antibiotics lead to the disassembly of ribosomes. One intriguing possibility is that scientists could develop antibiotics that target bacterial ribosomes specifically and interfere with their assembly, "essentially throwing sand into the gear box," says Williamson.

In one corner of Williamson's office is an old foam mouse pad adorned with the ribosome, a freebie produced to celebrate and promote the long-awaited publication of a high-resolution structure. It is not the purple blob of old but the atomic shape of post-1998.

Still, it sits unused on a corner table, the victim of better mouse technologies that use laser motion detectors instead of track balls.

There is still a long way to go with the ribosome, says Williamson.


Send comments to: jasonb@scripps.edu



Professor Jamie Williamson's interest in RNA-protein complexes has led him to study the basic workings of the ribosome and potential new therapies for AIDS.





Until a few years ago, our best images of the ribosome were low-resolution models such as these, which were inspired by wooden shapes models Jim Lake created in the 1970's based on electon microscope images.






Atomic resolution structure of the 30S ribosomal subunit. The RNA chain is red and the 20 protein chains are blue. Two views are shown, one from the perspective of the 50S subunit (inside), and the other from the perspective of the solvent (outside). The cleft at the middle of the structure is where messenger RNA passes through as it is decoded during protein synthesis.