Book “Genetic Detective. From ribosome research to the Nobel Prize ”

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Venkatraman “Wreaths” Ramakrishnan is an American and British biochemist, winner of the 2009 Nobel Prize in Chemistry, together with Thomas Steitz and Ada Yonat, “for studying the structure and functions of the ribosome.” Since 2015, President of the Royal Society, member of the US National Academy of Sciences.

Everyone knows about DNA, the molecule that stores our genes. But DNA is useless without a ribosome - a unique processor that decrypts the genetic code. It is the ribosome that is the engine of life. “The genetic detective. From the study of the ribosome to the Nobel Prize ”is a fascinating story about the discovery of its incredibly complex structure and the unraveling of the ancient mystery of life.

Excerpt. First crystals

Their success motivated others to join in such work. The Soviet government organized a large scientific center in the town of Pushchino. There were several well-funded research institutes, one of which was headed by a brilliant biochemist and ribosome specialist Alexander Sergeyevich Spirin. Like Wittmann, he led a large scientific department, where almost all aspects of ribosomes were studied. He was not as systematic as Wittmann, had a rich scientific imagination and was ready to publish bold ideas. In addition, Spirin showed himself to be a very independent person who did not bend to the authorities. So, once he was asked to sign a petition with the initiative to expel Andrei Sakharov, the famous nuclear physicist, dissident, creator of the Soviet hydrogen bomb, from the USSR Academy of Sciences.An open refusal to sign such a petition might have seemed a politically short-sighted act on the part of a prominent member of the Academy of Sciences, besides the director of a large research institute, so Spirin decided to go on a hunting trip to the forests adjacent to Pushchino for a short while.

One of his collaborators, Maria Borisovna Garber, led a small group that attempted to crystallize individual ribosome proteins or protein factors that provide various ribosome functions. Like the rest, she worked on E. coli material.

Garber changed her approach to the study of ribosomes in 1978 by reading one Japanese report that described work with a new thermophilic bacterium, on the material of which two important proteins that acted in the ribosome were crystallized. A little tautologically named Thermus thermophilus, it was discovered by Tyro Oshima in hot springs on the Izu Peninsula in 1971. It grows best at 75 ° C.

Garber traveled to Japan for several months and in December 1979 brought several such bacterial cultures to the USSR - but, unfortunately, microorganisms died on the way. She asked Oshima to send her fresh cells by mail, and they arrived safely. By the end of 1980, Garber and colleagues managed to fruitfully work on these bacteria and got beautiful crystals of protein (or rather, protein factor), called the elongation factor G, which helps the ribosome to move along the mRNA.

The first success with T. thermophilus proteins inspired Garber and her colleagues to experiment with this organism. It turned out to be quite expensive to grow T. thermophilus in the USSR, and Garber invited other Soviet scientists to disassemble the bacterium together for all proteins that could be useful.

Among his colleagues, Garber was Igor Nikolayevich Serdyuk, who was engaged in “contacts with foreign partners,” who often traveled to the West without problems even at the height of the Cold War. Previously, he applied low-resolution methods, outlining the shape of ribosomes, so naturally he wondered if they could crystallize using materials from the Garber lab. He and his student Elizabeth Karpova purified T. thermophilus ribosomes and obtained very small crystals, similar to the first crystals synthesized in Berlin. After this success, Garber turned to Spirin with a request to support the group and its attempts to crystallize the ribosomes of the new strain.

He agreed, and several more people joined the case, among whom should be noted Marat Yusupov, a student of Spirin. Having no serious experience in crystallization, scientists turned for help to two employees of the Moscow Institute of Crystallography - Vladimir Barynin and Sergey Trakhanov. By 1986, they were able to crystallize the small subunit, and also, using the technique by which Trakhanov purified ribosomes, a whole ribosome. Now, taking into account the 50S crystals obtained by the forces of Jonat and Wittmann, both subunits and the entire ribosome were at the disposal of scientists.

Yusupov presented his results in the form of a poster shown in July 1987 in Bishenbourg near Strasbourg in France, and a month later this work was published in the prestigious scientific journal FEBS Letters. After a couple of months, Jonat and Wittmann reported that they also managed to crystallize the small subunit and entire ribosomes on the material of the same T. thermophilus strain. They published their results in the same unknown journal Biochemistry International. The following year, Yonat wrote that they managed to improve the crystals of the small subunit (30S), and now they looked at least no worse than the Soviet ones.

This could lead to fierce competition between the Soviet and German groups, but this did not happen. The Russians received much less funding than the Germans, and were less equipped, especially for the crystallographic processing of large molecules. Trying to take the research to the next stage, Marat Yusupov and his wife Gulnara went to Strasbourg, where they were going to continue the crystallographic study of ribosomes with Jean-Pierre Ebel and Dino Moras. For reasons that remained unknown to Yusupov, Ebel at some point decided to stop cooperation. Spirin believed that Jonath and Wittmann convinced Ebel that it was not worth competing with them.

Whatever the real reasons, the Soviet developments on the crystallization of ribosomes have faded. Maria Garber returned to her former scientific interests: the study of individual ribosomal proteins and factors. Disappointed with the fruitless work, some key representatives of the Soviet research group dispersed around the world. A few years later, in the mid-1990s, Yusupov wrote to Harry Noller, a leading biochemist and ribosome specialist from the University of California, Santa Cruz, asking for permission to work on the ribosome structure in his laboratory, but this story should be left for later. Sergei Trakhanov led essentially a nomadic life, having managed to work in Japan and the USA for twenty years. He also worked for some time in the laboratory at Noller, after Yusupov departed from there; then Trakhanov returned to Europe.

With the actual closure of the Soviet project, Yonat remained at the head of the only ribosome crystallography group. By the end of the 1980s, it was not yet possible to obtain a single crystal so good that the atomic structure of both subunits of the ribosome, and even more so of the entire object, was visible in it. But they were quite suitable in order to roughly judge how proteins and RNA interact in the ribosome.

Indeed, the structure of the ribosome gradually formed into a map of blurry images obtained using an electron microscope. Part of the work that was relevant at that time was associated with antibodies, that is, with proteins that are synthesized by our immune system and can attach to strictly defined targets. In one experiment, Jim Lake of the University of California at Los Angeles synthesized antibodies that recognized the start of a newly made protein. In 1982, he demonstrated that these antibodies attach to the back of a large subunit, that is, exactly opposite the point where a new amino acid with tRNA attaches to an expandable polypeptide chain. The conclusion was that a large subunit should have a tunnel: a kind of birth canal, which will pass a new protein chain,before appearing on the other side. In 1986, Nigel Anwin confirmed the existence of such a tunnel by analyzing electron crystals of flat crystals obtained from lizard ribosomes using an electron microscope. The following year, Jonat and Wittmann also reported that there is a tunnel in the subunit, relying on the flat sections of the ribosome crystals that they themselves obtained using electron microscopy. Both of these reports were based on low-resolution images, far from the current view of the ribosome, but scientists confidently identified other objects as a tunnel, the existence of which had already been proved by Lake.The following year, Jonat and Wittmann also reported that there is a tunnel in the subunit, relying on the flat sections of the ribosome crystals that they themselves obtained using electron microscopy. Both of these reports were based on low-resolution images, far from the current view of the ribosome, but scientists confidently identified other objects as a tunnel, the existence of which had already been proved by Lake.The following year, Jonat and Wittmann also reported that there is a tunnel in the subunit, relying on the flat sections of the ribosome crystals that they themselves obtained using electron microscopy. Both of these reports were based on low-resolution images, far from the current view of the ribosome, but scientists confidently identified other objects as a tunnel, the existence of which had already been proved by Lake.

Apart from these results, progress has been slow. Even a decade after the first three-dimensional ribosome crystals were obtained, it remained unclear whether it would be possible to construct at least some intelligible maps based on X-ray crystallography. Nevertheless, Ada Yonat clung to this dream and sought to improve her crystals.

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