An eucariotic ribosome is a ribonucleoprotein complex consisting of 4 ribonucleic acids and about 80 proteins. The main function of ribosome is to serve as the site of mRNA translation into a sequence of amino acids. We observed dynamic changes of ribosome’s conformation during protein biosyntesis. These changes are correlated with the steps of the elongation cycle. Most important for understanding the translational machanism is how the ribosome interacts with the substrates and other important factors involved in polypeptide elongation. One of the methods allowing to investigate this complex mechanism is the antisense strategy. The main idea of this strategy is to introduce short antisense oligodeoksyribonucleotides (a-DNAs) complementary to important parts of 26S rRNA into the ribosome. The effect of the hybridization of the selected antisense oligomers has been analyzed after the ribosomal transition from pre- to posttranslocation state with tRNA and/or tree types of tRNA molecule lacking some crucial fragments (mini-tRNA). We believe that mini-tRNA could be used for identification and characterization of tRNA-rRNA interactions in the ribosome and could provide new information about interactions between tRNA and functionally important rRNA domains. Specificity of the a-DNA/ribosome interactions was assured using bioinformatics algorithms and the analysis of the free energy (ΔG) of RNA/DNA duplexes.

We noticed significant changes in the conformation of the ribosome as we observed molecular ratio of specyfic hybridized oligomer per ribosome.We concluded that the structure of the ribosome seems to be more open in pretranslocation state especially when a-DNA is hybridized to ribosomes occupied with mini-tRNA. We think, that according to smaller size of mini-tRNA (comparing to tRNA), more space in the ribosome is available for a-DNA. In the next step of experiments, we will perform inhibition of AA-tRNA binding to poly-U programmed ribosomes occupied with mini-tRNA or d-tRNA.

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1. Analysis of sites susceptible to N-homocysteinylation in human blood proteins
2. Monitoring the dynamics of translating ribosomes.
3. Identification of RNA motifs specifically binding human ribonuclease Dicer