29. augustil 2013. aastal kell 14.00 kaitseb Margus Leppik will defence his doctoral thesis "Substrate specificity of the multisite specific pseudouridine synthase RluD" on the 29 August at 14 pm in the Institute of Molecular and Cell Biology of the University of Tartu (Riia 23B-105) in the specialty of Molecular Biology.
Prof Jaanus Remme, Institute of Molecular and Cell Biology of the University of Tartu
Senior Research Fellow Aivar Liiv, Institute of Molecular and Cell Biology of the University of Tartu
Senior Lecturer Petr V. Sergiev, Faculty of Chemistry in the University of Moscow (Russian Federation)
One of the most important processes in every cell lifecycle is protein synthesis performed by ribosomes. Ribosomes are composed of two unequal subunits (large and small subunit). Small subunit is responsible for following the genetic code and large subunit is responsible for synthesis of proteins. Ribosome consists of ribosomal RNA (rRNA) and r-proteins. Structure of rRNA is very conserved and it carries the catalytic role in protein synthesis. rRNA contains many modified nucleotides which are also highly conserved. Although modified nucleotides are known for a long time, there is little information about their function in rRNA. The most common modifications in rRNA are pseudouridines which are synthesized by proteins called pseudouridine synthases. E.coli RluD is pseudouridine synthase that synthesizes three highly conserved pseudouridines located in a very important structural element of the ribosome, helix 69 (H69). Little is known about how RluD recognizes its substrate nucleotides, or why these modifications are needed. We used genetic manipulations to introduce site specific point mutations into rRNA and RluD genes in order to determine the effect of the mutations on the occurrence of RluD directed pseudouridines. Our results show that RluD is specific to positions 1911, 1915, and 1917 in fully assembled ribosomes. However, RluD exhibits loosened specificity on protein free rRNA. Our results suggest that RluD exhibits a two-step binding to its substrate. The S4-like domain of RluD is responsible for initial binding that guides the catalytic domain to the substrate. We found that the catalytic domain of the RluD probably contacts nucleotide at position 1916 of the 23S rRNA to achieve its specificity. We also found that all RluD directed pseudouridines occur concurrently and independent of each other to the rRNA. Our results help to better understand the mechanism of ribosomal biogenesis in cells.