30 years of research and one Nobel Prize: a conversation with Prof. Ada Yonat about the journey to decipher the structure of the ribosome
When we entered the office of Prof. Ada Yonath, winner of the 2009 Nobel Prize in Chemistry, she was busy packing one of the first models of the ribosome made in her laboratory, a model made of styrofoam built layer by layer, which she intended to send to the Nobel Museum in Stockholm, Sweden. The nesting pigeon through the model has a red string and a blue string, representing the mRNA and the newly formed protein chain, respectively.
The model will be displayed in the museum alongside exhibits such as Galileo's telescope, Yitzhak Bashevis-Singer's typewriter and more. But it seems that Yonath was more moved by sending letters of thanks to the children from whom she received blessings; And especially from a letter she wrote to the children of the third grade in Kiryat Tivon, who sent her a file of illustrated congratulations the day after the announcement of her win.
The wall to her right is paved with many certificates that she received for her achievements over the years, such as the Wolf Prize, the Israel Prize and also a certificate she received from her granddaughter: "The grandmother of the year is Ada Yonat". But the path that led to the recognition of her achievements and the various awards was not easy.
The diagram shows the ribosome, which consists of the small subunit and the large subunit when translating the genetic information from a messenger RNA molecule (mRNA) to build a protein.
to study the processes of life
Yonat was a scientist almost by accident. Although she admired Marie Curie as a child, also a winner of the Nobel Prize in Chemistry, she did not think that she would engage herself in chemistry in particular and science in general. Yonat remembers that the chemistry classes in high school, which she enjoyed very much, were conducted in a slightly less glamorous way: "The chemistry teacher called me a head of cabbage. In fact, the whole class was head of cabbage, the biggest committee", she recalls with a smile, "he really loved me, and especially loved me because he would say I was talkative and would take me out. So I would wash the dishes in the lab. He would enter the classroom and say: Today we will learn about salts, Ada - to the laboratory." Yonat adds and says: "I didn't know there was a profession called a scientist. I thought that you study science at university but then you go to work in all kinds of laboratories, hospitals, factories... I also knew that you had to study to be a high school teacher. While in the army, I studied in the evening to have a profession, and I was already a teacher in a public school - I knew that if I wanted to be a high school teacher, I needed another certificate. But I didn't know that there are people who come to work, do what interests them for fun, and also get paid for it."
At first Yonat wanted to go to work in the kibbutz out of a sense of Zionism, but she had to help support the family. Yonat's father died when she was a child and her mother and little sister needed her help. Yonat decided that she should turn in a different direction: "My mother lost all the family's capital after receiving the key fees for the apartment in Jerusalem, and intended to use the money to pay for the apartment in Tel Aviv. On the way, between the moment she boarded the bus in Jerusalem and the moment she got off in Tel Aviv, the great devaluation of 1952 took place and the value of money shrank by a third." Since she received many scholarships and at the same time worked small jobs in her spare time, it was her studies at the university that allowed her to support her family.
During her studies at the Hebrew University, Yonat navigated between her love for chemistry and that for physics, and the decision to study chemistry in her undergraduate studies was quite accidental. As part of her master's degree, biochemistry attracted her, with a specialization in biophysics: "I really wanted biochemistry, much more than the other chemistry - organic, inorganic. I was more interested in understanding the processes of life." But even at this stage she did not yet see herself as a scientist: "I liked it a lot, but even when I was doing my master's I didn't think of myself as a scientist or science as a profession. Until then, I knew [the scientists] as teachers... When I got here, to the Weizmann Institute, I said good, we'll do a doctorate, and that's how it continued." In her doctoral thesis, Yonat studied the protein collagen - the main component of the white fibers in the connective tissues, ligaments, tendons, skin, bones and cartilage.
After finishing her doctorate, Yonat went to postdoctoral studies abroad. When she returned to the Weizmann Institute in 1970, she established in the Faculty of Chemistry the first laboratory in Israel for the study of proteins using X-ray crystallography. When Yonat's appointment as a young scientist at the Weizmann Institute was about to end, the extension of the appointment and the continuation of her research work were in doubt. At that time, shortly after the Yom Kippur War, Yonat offered Rafael's people a computational method that was supposed to help improve the accuracy of the missiles. Raphael approached the Weizmann Institute in order to offer Yonet a permanent position. This appeal, says Yonat, was probably of considerable weight in extending her appointment for another two years. In this battle, rocket science lost to ribosome research.
In order to decipher the structure of a substance with the help of X-ray crystallography, one must first produce from it crystals that are as clean as possible. The crystals are irradiated with X-rays. The rays passing through the crystal are scattered in a phenomenon called diffraction. The resulting scattering pattern and information about the scattering directions allow deciphering the arrangement of the atoms in the crystal. The more perfect the crystal, the better resolution a map of its structure can be created, but obtaining such a crystal is not easy. The resulting scattering pattern and information about the scattering directions allow deciphering the arrangement of the atoms in the crystal. The more perfect the crystal, the better resolution (degree of separation) a map can be made of its structure, but obtaining such a crystal is not easy, especially when it is a very complex component like the ribosome. Indeed, the accepted assessment at the time was that it was not possible.
The journey to research the ribosome
Even as a student, Yonat felt that in order to understand the processes that take place in the living cell, the spatial structure of the participating components must be understood. "Life processes are very dependent on the space in which they are located. The arrangement in the space is the one that determines whether the process will be carried out or not, and how." When she arrived at the Weizmann Institute, Yonat sought to understand in this way one of the most important processes - RNA translation and protein creation. "The translation process, which has two languages - one of four letters, one of 20 - has a quick selection of who should enter and who should not, and also information about the folding of the protein - this is such a central process that practically every drop of information we put into it will be significant."
When Yonat began her studies, it was known that the ribosomes serve as the protein production factories of the cell and that they exist in a huge number of copies in every cell, in all living things from bacteria to oak trees. They are responsible for translating the genetic information stored in the nucleic acids and assembling a protein chain based on this information at a rapid rate of about 20 amino acids per second. The ribosomes consist of nucleic acids (rRNA) and many proteins organized as two subunits.
The large subunit, called 50S in bacteria, is about twice as large as the small subunit, called 30S. During the translation of the genetic code into a protein, the two subunits come together, like clam shells, around the mRNA to receive the complete ribosome (70S). But although an extensive study of the ribosome revealed many findings about its nature and its operation, without deciphering its spatial structure it was impossible to discover detailed information about its function.
However, the idea of deciphering such a complex structure of the ribosome seemed too far-fetched at this stage even for a pigeon, since ribosomes were considered impossible to synthesize due to their extremely large size, made of many proteins and RNA and lacking structural symmetry. Yonat joined Professor Michel Revel and his doctoral student, Yoram Groner, who were working with Professor Paul Sigler from Chicago who was then in Israel. Together they tried to decipher the structure of much smaller proteins, the initiation factors (factories) that participate in the protein creation process.
The work progressed and Yonat was able to obtain some small crystals - micro-crystals - of some of the proteins. "But then", she says, "Michel Rebel moved to interferon research and closed his bacteria laboratory. Yoram went to a post-doctorate and Professor Paul Sigler, who was the guest, returned to Chicago." Yonat was left without a source of proteins to continue her research. In those days, there was a separation between crystallography labs and "wet" labs where they grew bacteria and produced proteins, and Yonath depended on such a lab to supply her with raw materials. She followed Sigler to Chicago and began working there.
While she was with Sigler, Yonat flew to a conference in Canada, where she met Hans Guenther Wittmann, president of the Max Planck Institute for Molecular Genetics in Berlin. It took her three days to muster up the courage to approach him, but as soon as she did - she immediately received an offer from him to come and work with him. Yonat came to visit Whitman's laboratory and it was decided on a collaboration in which she and her students would work four months a year in Germany and in return would receive a third of the proteins that the laboratory would produce. But the plan did not come to fruition due to an accident in which Yonath fell off her bike and suffered a concussion. As a result, she had a lot of time to read, or in her own words: "I read about the bears and that's where it all started."
When Yonat returned to Whitman at the end of 1979, she told him what she had read: that the ribosomes of polar bears are packed in an orderly manner, that is, they crystallize, when they hibernate during the winter, and therefore it is possible, apparently, to synthesize ribosomes in the laboratory as well. Since there were many ribosomes at the Max Planck Institute where she worked, Yonat began to work on their synthesis and as early as 1980, she published, with Whitman and other colleagues, that she had succeeded in obtaining crystals of the large subunit (50S) of a thermophilic ("heat-loving") bacterium - a bacterium that lives at temperatures high
These crystals were a necessary first step in the journey that lasted about 20 years to decipher the structure of the ribosome. But few people believed that Yonath was capable of making this journey: "At the institute they somehow let me breathe. The Max Planck Centers were very, very interested and decided that the expenses on me were too small to argue. But in the world I would be considered as the dreamer, the delusional, the delusional, the wishful thinker, who does not know how to interpret results, who does not understand what she is doing, who has already reached a state of near-end and has not reached the end." But despite the criticism, Yonat had mental strength that allowed her to continue, since even if she didn't always believe that she would arrive at deciphering the structure of the ribosome, "I knew where I was going." I knew that the final goal was very high and that even if I reached half the height it would still be scientifically significant."
Yonat of course did not work alone all these years. There were students - undergraduates and doctoral students - who were excited enough by the possibility of deciphering such an important structure to come and work in the laboratory even when it was not at all clear whether the research would end with significant results. Dr. Anat Beshen, for example, worked with Yonat as a doctoral student at the Weizmann Institute, but since in the years she worked the laboratory was unable to decipher a structure, she almost did not receive the doctorate. Yonat had to explain that her work in the field of data analysis was critical to the research, even if there is still no beautiful picture of a ribosome to decorate her thesis. Today Bashan works with Yonat as the senior scientist in her laboratory.
The structure of the ribosome. You can see in the figure the small subunit (30S) and the large subunit (50S), three tRNA molecules located in the A, P and E sites.
Bacteria, crystals and the structure of the ribosome
One of the main problems was the fact that in order to extract information from the crystals they had to be stable enough to withstand strong X-radiation without disintegrating. Yonat chose to work with bacteria living in extreme conditions because she assumed that their ribosomes, which have evolved to withstand harsh conditions, are more stable than those of bacteria living in normal conditions. One of the bacteria she chose is the archaeon ("ancient bacteria") from the Dead Sea, Haloarcula marismortui. This archaeon is an extreme halophile ("salt lover"), meaning it must grow in extremely high concentrations of salts, and as a result its ribosomes are very stable. Indeed, although the archaeon's ribosomes also quickly disintegrated when exposed to radiation, they survived long enough to produce initial information about their structure. Yonat used in her work, in addition to the archaeon, also ribosomes produced from Thermus thermophilus, a bacterium ("real" bacterium) which is an extreme thermophile, as well as Deinococcus radiodurans which is a bacterium resistant to radiation, heat and dryness, yet it simulates pathogenic bacteria (see: Sparrow Bar- Nir, "On radiation and bacteria", "Galileo" 110).
Yonat overcame the rapid disintegration of the ribosome crystals due to their X-ray irradiation by rapidly cooling the crystals to about 180 degrees below zero, a method she developed with the help of Hakon Hope (Hope) from the University of California at Davis. Thanks to this method, cryo-crystallography, the ribosomes produced from the bacteria resistant to external conditions survived the exposure to radiation. Stabilizing the crystals made it possible to analyze them using a new technology of synchrotron radiation, focused and high-intensity X-rays, the difference between which and "normal" X-rays is like the difference between a laser and light from a normal flashlight. Yonat studied the crystals at synchrotron facilities around the world, such as the European Synchrotron Radiation Facility (ESRF) in Grenoble, to obtain increasingly high-resolution data on the structure of the ribosome.
In 1995 Yonat made another important innovation in ribosome research. She developed a method for marking the crystals to obtain reference points that facilitate deciphering the scattering pattern as a three-dimensional structure. Although the marking by heavy atoms is not new in the field of crystallography, Yonat used clusters of atoms of heavy metals to create a particularly prominent marking. This labeling is a real breakthrough, which actually made the existing information about the ribosome readable, and thus opened the door to studying the function of the ribosomes.
At these stages, when it was clear that the work was progressing towards deciphering the structure, colleagues who until not long ago regarded Yona as a dreamer and did not believe that she would succeed, joined the race and the competition to decipher the structure of the ribosome became tough. In front of Yonat's relatively small laboratory stood two much larger and more equipped laboratories of American researchers: Steitz from Yale University and Ramakrishnan who was working at the time in Cambridge, England. The researchers, who shared the Nobel Prize with Yonat this year, "jumped on the bandwagon" and tried to get ahead of Yonat in publishing the structure of the ribosome, while using the methods she developed. They even used bacteria living in extreme conditions that she chose. Steitz began studying the large subunit of H. marismortui, the Dead Sea archaeon, and Ramakrishnan the small subunit of T. thermophilus.
In 1999, the three laboratories published their research on the structure of the ribosome, with Yonath's laboratory publishing a structure of the small subunit with a relatively low resolution, but better than the competitors. A year later, three high-resolution structures were published within five weeks, allowing a three-dimensional model of the locations of the nucleotides and amino acids in the ribosome to be built: Steitz published in Science on August 15, Yonet in Cell on September 1, and Ramakrishnan in Nature on 21 of the same month.
All this work, and work published by the groups afterwards, added a lot of information about the function of the ribosome. It turned out, for example, that there are no proteins near the active site in the ribosome - that is, the rRNA is the one that carries out most of the ribosome's activity. In the large subunit of the ribosome, the amino acids join together to build the protein. This unit also includes a tunnel, which Yonat identified for the first time, that protects the chain of the newly constructed protein and enables its exit from the ribosome. Also, the large subunit serves as a binding site for proteins that participate in the processes of processing the resulting protein. The small subunit is responsible for starting and ending the protein production process and decoding the genetic code and preventing errors.
In the ribosome there are three tRNA binding sites marked with the letters A, P and E and present in both subunits. Initiation of the translation process occurs when the small subunit binds to the mRNA molecule at the initiation codon. The first tRNA molecule (with its amino acid) is also bound to this codon, after which the large subunit is also bound to obtain a complete ribosome to whose P site the first tRNA is bound. The boot process is mediated by boot factors. After the initialization, the elongation phase begins where the protein chain is built (you can see a good example of this process in the link to the video at the end of the article).
At this stage tRNA carrying an amino acid enters site A. The rRNA of the small subunit "checks" whether this is the correct tRNA. If so, then a movement occurs in the rRNA of the small subunit leading to a change in its spatial arrangement (conformation). At the same time, the part of the tRNA carrying the amino acid rotates in the direction where the connection between the amino acid bound to the tRNA at the A site and the nascent protein chain, which is meanwhile at the P site of the large subunit, can be formed. In the process, the tRNA that was in the A site moves in a movement consisting of straight displacement and rotation to the P site, while it is still bound to the amino acid and through it, now, to the entire protein chain. The tRNA that was previously in the P site separates from its amino acid, moves to the E site and is released from the ribosome.
The ribosome moves a distance of one codon along the translated mRNA, so that the A site again has a codon waiting for the appropriate tRNA. The elongation process is mediated by proteins called elongation factors, and it continues until one of the termination codons appears at site A. In this case, instead of tRNA, termination factors bind to site A, which release the protein chain from the tRNA at site P. The new protein leaves the ribosome, which returns and breaks down into the two subunits and is ready to start the translation process again.
Yonat developed a method for marking the crystals that would make it easier to decipher the scattering pattern as a three-dimensional structure, and used atomic clusters of heavy metals to create a particularly prominent marking.
Looking ahead
In researching the ribosome itself, deciphering the structure opened up possibilities that have not yet been fully exploited, such as the development of more effective types of antibiotics that are less likely to create resistant bacteria - one of the most difficult problems facing modern medicine.
Also, the structure of the nominal ribosome (of animals), which is somewhat different from the bacterial one, has not yet been deciphered. Even before synthesis, any attempt to obtain clean animal ribosomes encounters difficulties, because there are various particles and proteins that stick to them, even if the researchers use cells isolated in tissue cultures, which are cleaner than cells derived from living tissue. On top of that, animal ribosomes are even less stable than bacterial ribosomes, and it is even more difficult to synthesize them, or as Yonat says, "There is not a single animal that lives in the Dead Sea."
Yonat anticipates that the characterization with the help of a spatial structure will be extended to other processes besides translation. Many processes related to genetics and epigenetics are still waiting for this kind of decipherment. For example, the methylation process, in which a methyl group - a small chemical group - is attached to DNA. This process affects the ability of the gene to be transcribed and translated, therefore it is of great importance in controlling cell activity.
In the past, present or future, any scientific activity is always a group effort, and Yonat makes sure to thank everyone who works or has worked with her in the past. Everyone, she says, shares in the success - from her research colleagues to undergraduate students. "In the photos [published in the media after the win] I put all the people who donate. Let's say Dr. Tamar [Orbach-Nevo] who was with us until a year, a year and a half ago. We still post work with her that she did before and are still in touch with her. Officially she is not in the group, but I am grateful to her. Dr. Raz Zarivech, who is now a faculty member at Ben-Gurion University, and left us already four years ago: but he pulled us out of the mud, jump-started the project single-handedly. So I won't put him in the picture? Why, because he later went to Canada and is now in Beer Sheva? He is our child!”
Noam Levitan is a PhD student in life sciences. He holds a bachelor's degree in life sciences from the Open University and a master's degree in life sciences from the department of plant sciences at the Weizmann Institute of Science.
Yonat Ashhar has a certified degree in life sciences from the department of plant sciences at the Weizmann Institute and a bachelor's degree in life sciences from Ben-Gurion University.
Itamar Harel is a PhD student in life sciences at the Weizmann Institute of Science, researching the development and regeneration of facial muscles in the laboratory of Dr. Eldad Tzhor.
The article was published in Galileo magazine, February 2010
