Science has come a long way towards explaining the intricately woven fabrics of life. There is what Francis Crick called the central dogma: DNA makes RNA which makes protein which ultimately makes every living creature on Earth. At the dawn of the 20th century, the chemical foundations for life were mysterious. Since then, scientists have painstakingly chipped away at the mysteries of the central dogma, discovering the minute workings of complicated biological machinery. Examples include the 1962 Nobel prize awarded to Watson, Crick and Wilkins for discovering that DNA is a double helix, and the 2006 Nobel Prize to Roger Kornberg for figuring out the complexities of the transcription enzyme RNA polymerase. Now, the 2009 Nobel Prize in Chemistry is awarded to Venki Ramakrishnan of MRC Laboratory of Molecular Biology in UK, Thomas Steitz of Yale University in USA and Ada Yonath of Weizmann Institute of Science in Israel for the detailed mapping of the ribosome, a key player in the translation of RNA to protein. This discovery allowed us to glimpse a more complete picture of the behind-the-scenes complexity of the central dogma, and of life in general (1).
Background
Ribosomes, complexes made of ribosomal RNA (rRNA) and protein, are cellular components that carry out protein synthesis in all living cells, from bacteria to those of humans. Ribosomes translate the information in messenger RNA (mRNA) to produce protein and it is this stage that propels life into the realm of miraculous complexity. The body consists of tens of thousands of different proteins that control the body from hemoglobin that transports oxygen throughout the body to antibodies that fight intruding foreign particles.
Structurally, the bacterial ribosome consists of a small 30S subunit and a large 50S subunit, with respective molecular weights of about 800,000 and 1,500,000 Daltons (Da). The small subunit is comprised of around 20 different proteins and a ribosomal RNA (rRNA) of about 1600 nucleotides while the large subunit is comprised of around 33 different proteins and two rRNA sequences of around 2900 nucleotides and 120 nucleotides. Eukaryotic ribosomes are much larger in size and more complex than prokaryotic ones, but they all function according to the same basic principles.
The ribosome reads the nucleotides in the mRNA in codons (triplets). There are 64 different codons and 20 different amino acids, so some of the amino acids are coded by more than one codon, a property known as redundancy. Specifically, the ribosome has three binding sites for transfer RNA (tRNA), a small cloverleaf shaped RNA of 74-95 nucleotides, which brings the correct amino acid to the ribosome: the A (aminoacyl) site, the P (peptidyl) site and E (exit) site. The synthesis of a protein in bacteria starts when the mRNA binds to the ribosomal small subunit, which is followed by binding of the initiator tRNA carrying amino acid derivative formylated methione to the P site. The large subunit then docks to the small subunit and the complete ribosome with both its small and large subunits in place is formed (2). With mRNA in place, initiator tRNA in the P site and the empty A site programmed with the first codon of the protein to be synthesized, the ribosome leaves the initiation phase and enters the peptide elongation phase.
The 50S subunit of the ribosome. Proteins are in blue, RNA is in orange, and the active site in red.
In the elongation phase, the tRNAs enter the A site and peptide bond formation is catalyzed in the peptidyl-transfer center of the large subunit (3). An elongation factor then translocates the A-site bound tRNA to the P site and the P-site bound tRNA to the E site, moving the mRNA reading frame forward and programming the A site with the next codon to be read by the appropriate tRNA (4). The elongation process is repeated until the ribosome comes across a stop codon in the A site, which induces the hydrolysis of the ester bond that links a newly synthesized polypeptide with the P-site bound tRNA, thus leading to the release and folding of the polypeptide into functional protein (5). The ribosome is then recycled to a new round of initiation with a new mRNA.
Discovery
“Knowledge of a compound’s structure is absolutely essential in order to interpret its properties and reactions,” said Professor G. Hägg in his 1964 Chemistry Nobel Laureate lecture. Understanding the mechanisms of ribosomes is no different but the issue arose in early 20th century when ribosome crystallography was extremely difficult. The ribosome is not only large but also lack symmetry properties that would facilitate crystallization and structure determination, and it was unclear whether ribosome crystals diffracting to high resolution (< 3 Ångstrom) could ever be found. One Ångström (1 Å) equals one tenth of a millionth of a millimeter!
Ada Yonath came into the scene around this time, painstakingly trying to generate X-ray crystallographic structures of the ribosome, a feat which was at the time considered impossible. X-ray crystallography, which demystified the structure of DNA, is a method of determining the arrangement of atoms in a crystal in which a beam of X-rays strikes a crystal and diffracts into a specific pattern. Scientists can analyze this diffraction pattern and determine precisely how the atoms are positioned in a crystal. For x-ray crystallography to work, the crystal must be almost perfect where molecules repeat a precise pattern over and over again. Having the right condition is essential. It is very difficult to obtain high quality crystals from a protein and the larger the protein, the more difficult the task. Yonath’s vision is big. The ribosome is one of the most complex protein and RNA complexes with two subunits, each having thousands of nucleotides and thousands of amino acids, which in turn consist of hundreds of thousands of atoms. Yonath wanted to establish the exact location of each and every one of these atoms in the ribosome.
Yonath decided to work with Geobacillus stearothermophilus, bacteria that can live in warm springs as hot as 75 °C, since she reasoned that in order for the ribosome to still function under such high temperatures, it must be incredibly stable and hence would form better crystals. Yonath was able to generate the first three-dimensional crystals of ribosome’s large subunit in late 20th century and after 20 more years of hard work, she was able to generate a ribosome image where she could determine the location of each atom. She was a pioneer all right, from stabilizing the crystals by freezing them in liquid nitrogen at -196 °C to trying to crystallize ribosomes from other micro-organisms including the salt-loving Haloarcula marismortui. Soon, the word began to spread and more scientists joined the race including Thomas Steitz and Venkatraman Ramakrishnan.
Yonath’s crystals were good but not perfect. There was the “phase problem” of X-ray crystallography. Scientists needed the “phase angle” for every dot in the diffraction pattern in order to piece together a structure and to achieve this, they often soaked the crystal in heavy atoms such as mercury and by comparing dotted patterns from crystals with and without heavy atoms, the phase angle could be determined. Ribosomes, however, are too large with too many heavy atoms attached. It was Thomas Steitz who ultimately solved the problem using images of the ribosome generated via electron microscopy. These images helped Steitz figure out the orientation and location of ribosomes within the crystal. This was the missing link that together with heavy aheavy atom information gave the correct phase angle. In 1998, Steitz published his significant breakthrough.
Producing more and more crystals for better image resolution was all that remained to be done. In 2000, all three of this year’s Chemistry Nobel laureates published crystal structures with resolutions that allowed interpretation of the atomic locations (6). While Thomas Steitz obtained the structure of the large subunit from Haloarcula marismortui, Ada Yonath and Venkatraman Ramakrishnan obtained the structure of the small subunit from Thermus thermophilus. Once again, scientists did the impossible and mapped one of the most complex machineries in the cell, the ribosome.
Remaining the Fittest
Since World War II, the widespread introduction of antibiotics to treat bacterial infections has revolutionized medicine and dramatically improved global health. Six decades later, pathogens are fighting back and evolving antibiotic resistance. The ribosome is the target for around 50% of all current antibacterial drugs and the advent of high-resolution structures of both ribosomal subunits has given rise to opportunities for the development of effective drugs in the duel between bacterial pathogens and humans.
This year’s three Nobel Laureates in chemistry have all produced structures that show how different antibiotics bind to the ribosome. While some block the tunnel through which the growing proteins leave the ribosome, others prevent the formation of the peptide bond between amino acids. Several companies have already jumped at the opportunity to use these structures in developing new antibiotics with some of these new drugs already undergoing clinical trials. The discoveries that Ada Yonath, Thomas Steitz and Venkatraman Ramakrishnan have made, are essential both for deeper understanding of how life’s core processes function, and for Homo sapiens species to continually come out the winner as the jungle’s fittest survivor.
References
1. The Nobel Prize in Chemistry 2009: Scientific Background. Available at http://nobelprize.org/nobel_prizes/chemistry/laureates/2009/cheadv09.pdf (December 2, 2009)
2. A. Antoun, M.Y. Pavlov, M. Lovmar, M. Ehrenberg, The EMBO journal 25, 2539-2550 (2006).
3. M.V. Rodnina, M. Beringer, W. Wintermeyer, Trends in biochemical sciences. 32, 20-26 (2007).
4. J. Frank, H. Gao, J. Sengupta, N. Gao, D.J. Taylor, Proc. Natl. Acad. Sci. 104, 19671-19678 (2007).
5. L.L. Kisselev, R.H. Buckingham. Trends in biochemical sciences. 25, 561-566 (2000).
6. The Nobel Prize in Chemistry 2009: Presentation Speech. Available at http://nobelprize.org/nobel_prizes/chemistry/laureates/2009/presentation-speech.html (December 2, 2009)
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