Ribosomal proteins L5, L15 and elongation factor 2, three vital components of the translation machinery : Functional features of RPL5, RPL15 and EF2 from Saccharomyces cerevisiae studied in vivo

Sammanfattning: Protein synthesis is an essential, energy consuming and tightly regulated process in all living cells. A central core of the cellular protein-factory is a macromolecule called ribosome. Ribosomes are composed of ribosomal RNAs (rRNAs) and proteins (RPs). Additional components such as elongation factors (EFs) also contribute to this process. rRNAs are known as the catalytic constituents of the ribosome, while RPs are regarded as scaffold of the rRNA backbone. Despite this common view, in recent years, the functional importance of RPs has become more evident. In addition, RPs also carry extra-ribosomal functions some of which are linked to various diseases.In the current thesis I have attempted to highlight the importance of the structural features of two ribosomal proteins, YrpL5 and YrpL15A, present in the large ribosomal subunit and YeEF2. The results presented here are based on mutagenesis analysis, combined with functional complementation approach in the baker’s yeast S.cerevisiae.Introduced mutations show various degrees of cellular effects; YrpL15A tolerated inserted mutations greater than YrpL5. Nevertheless, YrpL15A proved to be more sensitive in its terminal-ends. This is presumably due to close contacts to the neighbouring molecules through these regions. On the other hand the N-terminal of YrpL5 displays a more permissive character for introduced mutations. In addition, A.thaliana orthologue to rpL15 could functionally substitute for yeast rpL15A. In contrast orthologues of rpL5 from A.thaliana, D.melanogaster and M. musculus were unable to functionally substitute for yeast rpL5. This could be an indication of species-specific features in YrpL5. Furthermore, two regulatory and highly conserved amino acids Thr56 and Thr58 displayed unessential functional role in yeast eEF2 under standard growth conditions. However, they showed to be important for YeEF2 function under mild osmotic stress. This may point to alternative regulatory mechanism for YeEF2.