Animation: John Rueter, Portland State University
RIBOSOMES, made up of two subunits (gray and violet), ride along threads of messenger or mRNA (yellow), translating genetic information (above). Codons--stretches of three bases--on the mRNA are matched up with complementary anticodons on transfer RNAs (green and pink), thereby forming the polypeptide chains (navy squares) used to build proteins. New detailed images of ribosomes (below) have revealed how the three binding sites for tRNAs (green, pink and blue) act as a conveyor belt, as well as other mechanical aspects of protein formation.

Image: CATE et. al.
The latest pictures of ribosomes may not be the most arresting biological images--but they represent a giant leap forward in the refinement of X-ray crystallography and in understanding a fundamental puzzle in molecular biology: how these tiny organelles, like highly efficient factories, manufacture all of the proteins for sustaining life from simple genetic templates. The new results, says researcher Jamie Cate of the Whitehead Institute "show where different pieces of equipment are on the shop floor."

Two papers in the September 24th issue of Science describe the images--the first to resolve an entire ribosome to 7.8 angstroms. The longer paper, by Harry F. Noller of the University of California, Santa Cruz, his colleagues Marat Yusupov and wife Gulnara Yusupova, Cate and Thomas Earnest of the Lawrence Berkeley National Laboratory, presents the structure of the ribosome and its interaction with several molecules. The second, by Noller, Yusupov, Yusopova, Cate and Gloria Culver of Iowa State University, details connections within the ribosome.

These reports follow two others in Science and Nature in August, which outlined different aspects of the ribosome's structure. But this recent flurry of results comes after some three decades of little progress. The ribosome--three RNA strands and 54 proteins woven into two separate, but entangled lumps--has not been an easy knot to unravel; its form has proved as hard to figure out as its function.

Ada Yonath of the Weizmann Institute of Science and Max Plank Institute for Molecular Genetics, who also has new results yet to be published, began trying to create images of the ribosomes structure in the late 1970s. She revealed her first success at crystallizing the organelle and using X-ray diffraction to produce an image at a meeting in 1980--pictures which garnered little enthusiasm from colleagues. Still, a few prominent mentors, such as Nobelist Sir John Kendrew, encouraged her, and in 1981, she made crystals that produced diffraction patterns clear enough to distinguish atoms in parts of the structure that were only 3 angstroms apart.

Achieving the same resolution for the entire ribosome presented new problems. But following Yonath's lead, other researchers were confident it could be done. Among them were Yusupov and Yusupova, who met as graduate students at the Protein Research Institute in Pushchino, Russia. Together they generated ribosome crystals by 1987, but did not have access to X-ray beamlines. So in 1996 they joined forces with Noller, who had been studying the biochemistry of ribosomes for 30 years, at UCSC. The team recruited crystallographer Cate and enlisted the help of Earnest, director of the crystallography facility at LBNL--one of the few places offering a synchrotron capable of producing X-rays with enough energy to create images of something as large as a ribosome.

In fact, the ribosome--measuring more than 200 angstroms in every direction--is the largest assymetrical object resolved using X-ray crystallography to date. Researchers have used the technique to produce images of viruses approximately the same size, but in these cases, they have recorded only part of the virus and taken advantage of its symmetry to generate the whole picture. To simplify their task, Noller's group used ribosomes from the bacteria Thermus thermophilus, which are slightly smaller than those found in higher organisms. And they used a cryo-electron microscope to first generate lower resolution images, which were then refined by diffracting X-rays of different wavelengths through crystals tagged with heavy atoms--a tactic called multiwavelength anomalous dispersion. That information, in turn, they used to pick out single heavy atoms in other ribosome crystals.

The resulting resolution was not sharp enough to define individual atoms, but it was better than they had hoped and revealed a number of the ribosome's secrets. In particular, the scientists were able to see how transfer RNA positions itself sequentially in three binding sites between the ribosome's two subunits during protein synthesis (see animation and image). "The transfer RNAs come through as if on a conveyor belt," Cate notes, "and we can see how the ribosome holds the transfer RNA differently in each of the binding sites." In one site, six "fingers" of electron density, like a robotic hand, tweak the incoming transfer RNA. "The ribosome appears to be a dynamic molecular machine with moving parts and a very complicated mechanism of action," Noller adds.

The images also made it possible to see more clearly how the ribosome's two separate units are tied together. "The most striking feature," the researchers wrote, "joining the two is an RNA helix." Some 100 angstroms long, this coil lies predominantly in the smaller subunit, but about once every helical turn touches the larger subunit in a loose stitch. Another thread of RNA from the larger subunit interacts with a protein component on the smaller unit. "Understanding how the subunits interact," Culver points out, "is critical to understanding how the ribosome works.

And that is the real goal. Given an atomic model of the ribosome, scientists could work out its structure at different stages of protein synthesis. Because antibiotics often act by disrupting bacterial ribosomes, such a model might very well pay out in the form of more specific drugs. "What we have at present," Noller explains, "are a few snapshots, and ultimately what we would like is a movie of the ribosome in action." At the rate his group has been working, it may soon be released.