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Structural biologists provides a close look at ribosomes

X-ray crystallography determines the position, interaction of individual atoms



This ribosome structure, determined at Argonne's Structural Biology Center, provides insight into every cell's protein construction zone. University of California, Santa Cruz image.
Evidence of human construction work is all around us: on the roads we travel, in the buildings we occupy and now even in outer space. But one form of “human construction” quietly does its job without making the daily news or creating the slightest bit of noise or inconvenience, unless its machinery goes highly awry.

This is the construction that has gone on inside every human cell – and probably every cell that has ever existed – since life began on Earth 3.5 billion years ago. Right now these cellular construction workers, called ribosomes, are at work in your eyes’ retinal cells, producing the molecules that allow you to see this page.

Ribosomes are composed of many separate molecules that work together to manufacture a cell’s proteins – large molecules that give our cells structure and initiate and speed up chemical reactions. Biologists working at Argonne’s Structural Biology Center (SBC) recently examined components of these protein factories with X-ray crystallography at resolutions high enough to determine the position and interaction of individual atoms. These images are the culmination of four decades of work in elucidating how the ribosome creates proteins.

Researchers have discovered that the ribosome’s basic mechanisms for producing proteins are probably billions of years old because parts of these mechanisms are preserved among all organisms, from humans to bacteria. Knowledge of the ribosome’s structure is also helping scientists understand how many antibiotics attack certain parts, or functions, of the bacterial ribosome. Pharmaceutical and biotechnology companies can use this valuable information to develop new antibiotics to fight the growing problem of bacterial drug resistance.

X-Ray crystallography at the APS

Three groups of researchers used the SBC beamlines at Argonne’s Advanced Photon Source (APS) to study ribosomes. The APS is a synchrotron that supplies the nation’s most brilliant X-rays for scientific research. The SBC beamlines’ brilliance enables the collection of atomic-scale structural data faster than at any other biological research facility in the United States. Researchers used the SBC to create complete, high-resolution models of the bacterial ribosome’s two main structural parts: the small 30S subunit and the large 50S subunit.

Each biology team directed brilliant X-ray beams from the APS synchrotron onto crystallized samples of the subunits. X-rays are uniquely suited for studying molecular structures because the wavelength at which the rays propagate through space matches the distances that separate individual atoms in a molecule. At the X-ray wavelength, photons – the light particles that compose the electromagnetic spectrum, including visible light – scatter, or diffract, off the negatively charged electrons in each atom of a crystallized molecule. The scattered photons hit electronic detectors that allow researchers to determine a molecule’s structure from the intensity and position of each scattered photon.

“Only researchers who come to third-generation synchrotrons with undulator sources, such as the APS, are able to obtain sufficient X-ray flux and brilliance to observe the reflections from ribosome crystals to atomic resolution,” said SBC director Andrzej Joachimiak.

The biologists faced a unique challenge in trying to crystallize the ribosome’s two subunits. Crystallization is normally the most difficult part of X-ray crystallography because of the varying conditions – temperature, acidity or alkalinity and concentration – in which molecules will crystallize. Each ribosomal subunit was several times larger than the next most complicated biological macromolecule whose structure had been solved at atomic resolution.

A macromolecule’s size and flexibility often make it difficult to form into the ordered, crystalline structure required for X-ray crystallography. But after much trial and error, the biologists found perfect conditions for crystal growth, such that each ribosomal subunit was packed identically into a single crystal containing trillions of identical units.

RNA is the ribosome’s tool of choice

The ribosome is made up of two subunits – the large and the small subunit. A Yale University research group, headed by Thomas Steitz, determined the structure of the large (50S) subunit at the SBC. This subunit is composed primarily of two long RNA molecules, a genetic material similar to DNA and found in all cells and some viruses.

The Steitz group found that the large subunit’s 31 proteins surround and stabilize the RNA, but do not contact the subunit’s catalytic site. These results showed for the first time that RNA performs the subunit’s main function – forming the peptide bonds between the amino acid building blocks in the chemical synthesis of a protein. Scientists can use this information to improve antibiotics that are already known to bind to the 50S subunit or design new ones that attack and bind to it.

Many biologists believe that in the early days of the Earth, when life was beginning to emerge 3.5 billion to 4 billion years ago, RNA was both the primary genetic material and the main catalytic substance that sped up the first protein-synthesizing reactions (see sidebar, page 14). Today, DNA is the primary genetic material, but RNA still is critical for making proteins by way of the ribosome. The process is now more complicated and precise than in its early history.

“RNA had to precede the creation of proteins because we know that the RNA component of the ribosome drives the formation of proteins,” said Steitz, the Eugene Higgins Professor of Molecular Biophysics and Biochemistry and Chemistry at Yale University.

Two separate groups resolved the structure of the small (30S) subunit at atomic-scale resolution. Ada Yonath led a group from the Weizmann Institute in Israel and the Max Planck Institute in Germany. The second group was led by Venki Ramakrishnan at the Medical Research Council (MRC) Laboratory of Molecular Biology in the United Kingdom.

While determining the structure of the small subunit, Yonath’s and Ramakrish-nan’s group discovered that ribosomal RNA is also the main player in decoding the genetic information inherent in a molecule called messenger RNA (mRNA), which is a transcripted copy of a gene’s instructions for making a protein. The small subunit contains one RNA molecule and 19 proteins, which support the RNA, much as they do in the large subunit.

“RNA’s function in decoding supports the idea that the ribosome is primarily an RNA machine and complements studies on the 50S subunit by the group at Yale,” said Ramakrishnan, lead of the structural studies division of MRC’s Laboratory of Molecular Biology.

The war on bacterial resistance

Ramakrishnan’s team grew three different crystals, each bound with a specific antibiotic to help determine how the small 30S subunit operated. Each type of antibiotic gave the structural biology group new insight into how the small subunit processes the information encoded in mRNA.

By blocking a specific function of the small subunit, each antibiotic gave the biologists not only a view of what happens when the process of translation – the creation of proteins from mRNA instructions – goes haywire, but also knowledge of how it normally proceeds.

“Scientists will now design better antibiotics by learning how the drugs bind to the ribosome and how structural and chemical changes can make their actions more specific,” said Joachimiak.

For example, Ramakrishnan’s group found that spectinomycin, an antibiotic normally used to treat gonorrhea, halts the growth of a nascent protein and keeps it attached to the ribosome’s small subunit. Antibiotic activity such as this will kill a bacterium because it can no longer manufacture new proteins.

“Pharmaceutical and biotech companies are keenly interested because this research not only helps us to understand how many known antibiotics work but also helps us to understand the basis of certain kinds of bacterial resistance,” said Ramakrishnan. “This will hopefully allow us to design new antibiotics in the future that can overcome the growing world-wide problem of resistance.”

The road ahead

Just as a detective must put together clues to a crime to identify the perpetrator, biologists have pieced together the operations of the ribosome’s subunits and built a near-final model of its structure and function. Progress in detective work and in structural biology only comes through refining methods and learning from past errors and successes.

Argonne will continue to contribute to this progression by supplying the technology and the know-how for important research on ribosomal structure. But down the road, instead of making crystals of just single subunits, biologists will create crystals of whole and mutant, or genetically altered, ribosomes at various stages in the process of translation to obtain a complete picture of life’s construction workers.

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For more information, please contact Evelyn Brown (630/252-5510 or eabrown@anl.gov) at Argonne.

 

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