Mechanism of protein biosynthesis in prokaryotes and eukaryotes studied by X-ray analysis – SSOTR

X-ray crystallography is an experimental technique of determining the arrangement of atoms within crystals, in which beam of X-ray strikes a crystal and causes the beam of light to spread into many specific directions. Based on the diffraction pattern obtained from X-ray scattering off the periodic assembly of atoms in the crystal, the electron density can be reconstructed. As result of subsequent treatment three-dimensional structure of macromolecule can be produced.
However, the main bottleneck in crystallographic studies is that a well-diffracting crystal must be created, and that the information gleaned about the dynamic nature of the molecules to be studied will be very limited from only a single diffraction experiment. In other words, the price to pay for the high accuracy of X-ray crystallographic structures is that the method is very time-consuming.
The main target of our project is the ribosome, which present the biggest macromolecules in the cell. This ribosome must be crystallized before its structure can be solved by X-Ray crystallography, and for many years there were no ribosome crystals with diffracting abilities. We were the first who solved structure of the bacterial ribosome in its functional state. Preparation of each new type of the crystal (for example ribosome with new drug, other functional ligand) suitable for X-ray crystallography can take several months, sometimes years. A further complication arises since ribosome crystals, as typically seen in RNA crystallography, diffract only poorly which results in electron density maps that are imprecise and difficult to interpret. Therefore special care has to be taken during post-crystallization treatment to avoid damaging the crystals and even for the freezing process itself we could only use the most robust methods.

For the ribosome, being a huge complex consisting more than 150 000 atoms, it is very important to make sure that the samples are homogenous for crystallization to succeed. Another important requirement is that the ribosome will be functionally active. By other words, we need to obtained snapshots of functioning of the ribosome at different stage of synthesis of protein, where the ribosome will be in complex with different players of biosynthesis, as for example messenger RNA (where information about future protein is encoded).
Other important requirement is high yield of purified ribosome, which will be necessary for the large-scale search of crystallization trials. Currently, we are developing the method of purification of functionally active ribosome from eukaryotes (cells with the nucleus, within which the genetic material is carried; human cells belong to the kingdom of high eukaryotes).
We are also developing model of functional ribosome complex, which will bring us to the first structure of eukaryotic ribosome functional complex.

The majority of antibiotics that inhibit bacterial translation target the ribosome complex, arresting translation at various stages, spanning initiation, elongation, termination, and recycling.
Comparison of the different drugs binding pockets between bacterial and eukaryotic ribosome on atomic level will give invaluable insight into the development of drugs against viruses, protozoa, fungi and bacteria.
Elucidation of the mechanism of biosynthesis of protein on ribosome will bring us the understanding of the molecular mechanism, for example, aging, or disease connected to appearance of aborted (not correct) proteins.

In the frame of the project 9 publications are published.

The ribosome is a giant ribonucleoprotein cellular assembly (molecular weight from 2x106 to 4.5x106 Da) that translates genetic code into protein in living cells. It is known that alterations in diverse range of translational machinery cause an extensive and growing catalogue of human diseases. For example, ribosomal protein defects have been associated with variety of blood and connective tissue disorders, including Diamond-Blackfan anemia, and rRNA pseudouridylation defects have been shown to be associated with dyskeratosis congenital.
Thus, the study of ribosome is important not only for the fundamental knowledge, but also for developing new therapeutic, which will target the ribosome. A complete understanding of protein synthesis requires knowledge of the structure of functional complexes of the whole ribosome with functional ligands such as complete transfer RNAs, messenger RNA, factors of translation, in its true biological context.
Presently only one technique can conceivably provide the high-resolution structural information the ribosome field ultimately requires: X-ray crystallography. Recent crystal structures of the prokaryotic ribosomal subunits, 70S ribosome from Thermus thermophilus and 70S ribosomal functional complexes provided new information about the functional surface of the ribosome, but nevertheless experimental visualization of the various steps involved in the process of translation is not complete. We have been working with the prokaryote T. thermophilus since 1993, and we were the first group to introduce this extreme thermophile in the field of ribosome crystallography, and obtain the first diffracting crystals of 30S and 70S from T. thermophilus. Our well-established protocols for structure determination of prokaryotic T. thermophilus 70S ribosome complexes provide us with an excellent platform for continued studies of the bacterial protein biosynthesis machinery.
Until recently no structure of eukaryotic ribosome (or ribosome subunit) was available. This year we determine first structure of the eukaryotic full 80S ribosome from Saccharomyces cerevisiae.
The overall goal of our research is to understand how the atomic structure of the ribosome ultimately determines its function. Towards this end we are using a combination of biochemical, biophysical and molecular genetic methods directed at a model prokaryote (Thermus thermophilus) and eukaryote organism (the yeast Saccharomyces serevisiae).