Leader: Olga A. Dontsova
Protein synthesis is one of the most important process in cell. Ribosome is a huge complicated complex consisting of RNA and proteins. Ribosome prepares the protein synthesis from the amino acids using the information coded in matrix RNA. Bacterial ribosome consists from two subunits – small (30S) and big (50S).
One of the tasks is to study the process of translation initiation and the role of initiation codons. mRNA includes 5’-noncoding region with Shine-Dalgarno sequence, which responsible for the right ribosome position. So, different variants of the Shine-Dalgarno sequence influence on the regulation of the protein translation. Furthermore, translation is impossible without the initiation factors, the role of them is not describe in full.
Other problem is the detection and research the rRNA and protein modifications during translation. It is known the big variety of various additional chemical functional groups which join to nucleotides and amino acids by specific modification in the cell. In bacteria very often separate enzyme modifys one position, so for cell it is necessary to synthesize a set of enzymes. Why is it so important for cell? What is the role of each modification? In ribosomal RNA there are more than thirty modified nucleotides, the most part is methylated. The majority of methylated nucleotides are in proximity from functionally significant ribosome centers and influence on the efficiency and accuracy of translation stages. Our interest is a concrete role of some modified nucleotides of rRNA and ribosomal proteins. For example, protein S6 contains two and more glutamic acids on the S-end, which considerably strengthen its total negative charge. Work is directed on finding-out how and during what moment and why for it occurs.
The third direction of the work in our group is studying of the antibiotics acting on the ribosome (3). Antibiotics suppress the bacteria growth or leads to the death. The majority of antibiotics block the protein synthesis in bacteria. It is known that bacteria are capable to adapt to the pernicious action of such substances, getting to them stability. The given phenomenon causes the requirement of search all new and new antibiotics. It is necessary to investigate mechanisms of their action for improvement of their antibacterial activity.
(ii) Studying of the telomerase in various organismsEukaryotic chromosomes end with special DNA-protein structures which are called telomeres. After being replicated telomere DNA shortens; this process leads to the genome instability and causes senescence. Telomerase is a unique enzyme, which is capable of synthesizing telomeric DNA on the basis of its own RNA subunit; the main function of the complex is to maintain telomere length. In humans telomerase is highly active in the cells which possess unlimited proliferative potential – they are known to be germ and stem cells. However, in most of the human cells telomerase activity is downregulated. Study of the telomere biogenesis allows to propose the connection between the low activity of the enzyme and aging of an organism. On the other hand telomerase upregulation is a key step for a cell to transform into a cancer one. It makes the complex an attractive target for the development of anti-cancer therapies.
The research in our laboratory focuses on several directions. We study structure and function of telomerase using yeast as a model system. We are especially interested in the Est3 protein, which is known to be important participant of S.cerevisiae telomerase complex. Its novel functions were shown in our lab, in particular, the ability to unbind specifically DNA-RNA hetero duplexes. This work enables to hypothesize that the Est3 plays an important role in providing processivity of the telomerase. Study of the Est3 regulation, its interaction with the telomeric DNA and telomerase will add much to our understanding of the telomerase action at the molecular level.
We also carry out the research which is aimed to study telomerase in higher eukaryotic organisms, human telomerase in particular. There are several questions which are addressed in the laboratory: regulation of the expression of the main telomerase components in human cells, telomerase RNA biogenesis and telomerase alternative functions which are not connected with the telomeric DNA synthesis.
Another direction includes search and creation of telomerase inhibitors and activators. This work is being done in collaboration with the department of Organic chemistry of Lomonosov MSU.
Research resultThe new class of compounds - inhibitors of telomerase was established, it’s an interesting as a potential basis for specific anticancer therapy. [Zvereva M.E. et al, 2011, WIPO Patent Application : WO/2011/126409].
The telomerase components of thermotolerant yeast H. polymorpha were identified, the components were isolated in pure and stable condition, which is the base for creating the telomerase system reconstruction. [Smekalova E. M. et al, 2012, Acta Natura].
The mechanisms of telomerase activity and telomeres length regulation were established for H. sapiens, H. polymorpha, S. cerevisiae. [Rubtsova M.P et al, 2009, Biochemistry (Mosc)].
Telomerase is active as monomer in vitro and biotinylated protein is a component of active telomerase complex in S. Cerevisiae yeasts. [ Shcherbakova D.M. et al, 2009, Biochemistry (Mosc); Shcherbakova D. M. Et al, 2009, Acta Naturae].
Biochemical properties and relations of additional protein Est3 S. cerevisiae from the telomerase complex were identify: GTP hydrolysis, specific cooperation with DNA and RNA heteroduplexes and quadruplexes in vitro. [ Shubernetskaya O. et al, 2011, Biochimie; Sharanov Y.S., 2006, FEBS Letters; Malyavko A.G., 2010, Dokl Biochem Biophys].
The special testing system for antibacterial products was investigated. The system is based on the reporter constructs with dual-fluorescent-protein genes. [Osterman I. A., 2012, Antimicrob Agents Chemother].
The number of report constructs (more than 100) were synthesized, in which one of the fluorescent proteins has different 5’- non-coding region, other protein is the control. This system can be very useful for the studying of the translation initiation and reinitiation. [ Sergiev P. V.,2012, Nucleic Acids Res.].
The method of purification of 30S ribosomal subunit by streptavidin affinity chromatography was established. [ Golovina A. Y., 2010, Biochimie].
It was shown, that C967 and G966 modifications in 16S rRNA help for the E.coli cells adaptaion to the bad conditions. The methyltransferase RsmD methylated G99 16S rRNA was investigated and described. [ Sergeeva O. V., 2012, RNA].
The new method of the modification searching was suggested. The basis of the method is the measuring the melting temperature of duplexes from rRNA and labeled DNA-oligonucleotides, which are complementary to the rRNA in the modification position. The first oligonucleotide has the FAM label, second – the BHQ1 quencher, so the building-up of the fluorescence can be fixed. The m7G and m6A modifications can be determined using this method.
The SAM-dependent methyltransferase YfiC was identified in E.coli cells. It methylates the N6 atom of the A37 nucleotide tRNAVal. It was shown, that this modification can help cells to survive under the osmotic and oxidation conditions. [Golovina A. Y., 2009, RNA].
Max Plank Institute of Biophysical Chemistry, Germany;
Max Planck Institute for Molecular Genetics, Germany;
Institute of Biotechnology, Lithuania;
EMBL, Germany.