In the office of Thomas Steitz, Sterling Professor of Molecular Biophysics and Biochemistry and Professor of Chemistry, there are numerous small models of each of the various macromolecular machines that his laboratory has “solved.”
It is an impressive collection that consists of an HIV-1 reverse transcriptase, a set of DNA polymerases in various states along the DNA replication pathway, a pyrrolysyl-tRNA synthetase, and a ribosome.
“My laboratory has been interested in understanding the mechanisms of the molecular machinery involved in the Central Dogma,” noted Steitz. “We want to make connections between structure and function.”
The Central Dogma of Biology tells us that DNA is transcribed to make a type of RNA, which in turn provides the molecular template to makes proteins, a process termed translation. However, many of the details of that big-picture flow remain to be understood.
During the past several decades, the Steitz laboratory has made considerable advances in elucidating the mechanisms of DNA replication, transcription, and translation. By selecting to study each molecule individually, atomics maps of a variety of macromolecules have been determined.
Many of these advances have led to new rational-based approaches for drug design as well as insight into fundamental pathways in life. Collectively, these results have allowed researchers to understand life at an unprecedented level.
One of Steitz’s most significant achievements came in 2000, when he published a high-resolution structure of the large subunit of the ribosome, the protein synthesis machinery of both prokaryotic and eukaryotic cells. This accomplishment revealed a multitude of details regarding the fundamental macromolecule.
Among these details was the demonstration that the active site of the ribosome is composed solely of ribonucleic acid (RNA). This observation had groundbreaking implications in understanding the molecular evolution of life.
Recently Steitz, along with Venkatraman Ramakrishnan from the United Kingdom and Ada E. Yonath from Israel, was awarded the 2009 Nobel Prize in Chemistry for “studies of the structure and function of the ribosome.” In their press releases, the Nobel Prize Committee notes both the scientific and clinical applications of Steitz’ achievements. The 1.4 million dollar prize was given at the annual awards ceremony in Stockholm, Sweden.
“It is an honor to receive the recognition of the international science community,” noted Steitz as he stared at a pile of unopened congratulatory letters and cards resting on his office desk.
By employing a powerful technique in structural biophysics, X-ray crystallography, Steitz, Ramakrishnan, and Yonath were able to position each of the atoms of the ribosome, a molecule that is approximately 2.5 x 106 Daltons in mass and 200 angstroms in diameter. The three researchers published the crystal structure independently of one other.
X-ray crystallography is one of the most fundamental yet powerful techniques for solving the structure of macromolecules. At present, it is the one possible way of studying the position of individual atoms.
In X-ray crystallography, researchers purify a macromolecule using a variety of column chromatography techniques (such as gel-filtration or ion-exchange). Once a sample is sufficiently pure, it is added in various concentrations to a variety of buffer solutions in an attempt to order the molecules and form well-defined protein crystals. This particular process is laborious and, in many circumstances, may take years to perfect.
Once a crystal is formed, it is taken to a particular type of particle accelerator known as a synchrotron, where it is bombarded with high-energy X-rays. The X-rays hit the atoms within the crystal and produce a diffraction pattern, which represents the positions of individual atoms. After analysis of a multitude of diffraction patterns, a final structure is modeled using crystallography software and rigorously examined to ensure that it accurately represents the in vivo structure.
Using a process similar to that outlined above, Steitz was able to solve the structure of the ribosome. However, given the size and complexity of the ribosome, numerous technical difficulties were presented, which Steitz had to overcome before completely solving the structure.
Peter Moore, Sterling Professor of Chemistry, Professor of Molecular Biophysics and Biochemistry, and a long-time collaborator and friend of Steitz, notes that once ribosome crystals were formed, it was relatively simple to measure the diffraction pattern of the macromolecule. However, determining the relative phases of each of the thousands of reflections contained was a difficult task.
The “phase problem” as it has been called in X-ray crystallography is a loss of phase information when taking physical measurements. The light detectors that are used in diffraction experiments collect only the intensity of the light. Since wavelengths contain both amplitudeand phase a crucial piece of information is lost. This information must be reconstituted to effectively determine the electron density map of a macromolecule, in this case the ribosome.
To circumvent the phase challenge, Steitz modified and then applied an approach Max Perutz devised to solve structures of macromolecules. He utilized a large multi-atom tungsten cluster to serve as a “reference point.” This cluster would bind with the ribosome and diffract as a distinct reference spot. This reference information allowed Steitz to form a complete data once multiple partial sets were integrated.
An additional problem rests within the limitations of computing software. As Steitz states, “The computer technology used in solving the ribosome was not available until the 1990s. It would have been impossible to accomplish the task before then.”
Overall, Steitz had to employ familiar techniques in a novel fashion.” Once these problems were solved, solving the structure of the ribosome was relatively straight-forward,” noted Steitz. Utilizing the complete diffraction data, which contained the phase information, Steitz performed a Fourier synthesis to determine the electron density of the ribosome and subsequently, its structure.
Today the Steitz laboratory is tackling a plethora of new questions in molecular biology. For instance, his laboratory has recently solved many structures of the ribosome complexed with a variety of antibiotics to provide a textbook for ribosome-targeted, rational-based drug design. Already, these structural insights are being successfully employed at Rib-X Pharmaceuticals, a company both Steitz and Moore help establish in New Haven, to design a new generation of effective antibiotics. Furthermore, Steitz is investigating the various states of the ribosome during the translation pathway. These studies have the potential for profound effects in medicine and pharmaceuticals.
Peter Moore remarked, “By understanding how various antibiotics target the ribosome, you can learn how to develop entire new classes of antibiotics.”
The structure of the ribosome for Steitz is not the conclusion of a journey, but the beginning of an exciting one. “For every answered question,” he said, “ten more emerge. No matter how much one pathway or process is studied, there will always be questions left unanswered.”
About the Author
PHONG LEE is a junior in Branford College majoring in Molecular Biophysics and Biochemistry. He is currently working in Professor Yorgo E. Modis’s laboratory studying both the structural mechanism of innate immunity and the membrane fusion of the Hepatitis-G virus.
The author would like to thank Professor Thomas Steitz, Professor Peter Moore, and Ms. Peggy A. Eatherton for their assistance with the article.