| 1. |
- Alkemar, Gunnar, 1970-
(författare)
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Ribosome and ribosomal RNA Structure : An experimental and computational analysis of expansion segments in eukaryotic ribosomal RNA
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ribosomes are large ribonucleoprotein complexes which incorporate amino acids into peptide chains during translational process in all types of living cells. The eukaryotic ribosome is larger compared to its prokaryotic counterpart. The size differences are due to a larger protein part and that the rRNA contains eukaryote specific expansion segments (ES). Cryo-EM reconstruction has visualized many ES on the ribosomal surface which have given clues about function and structural features. However, the secondary structures of most ES are unknown or ill defined. In this thesis, the secondary and also to a certain extent the tertiary structures of several ES are determined by using computational methods and biochemical experimental techniques. The juxtaposition of ES6 close to ES3 in the Cryo-EM image of the yeast ribosome suggested that ES3 and ES6 might interact. A computational analysis of more than 2900 sequences shows that a complementary helical region of seven to nine contiguous base pairs can form between ES3 and ES6 in almost all analyzed sequences. Biochemical in situ experiments support the proposed interaction. Secondary structure models are presented for ES3 and ES6 in 18S rRNA and ES39 in 28S rRNA, where homologous structural elements could be modeled in the experimentally analyzed ribosomes from fungi, plants and mammals. The structure models were further supported by computational methods where the ES6 structure and the ES39 structure could be formed in more than 6000 and 900 sequences respectively. A tertiary structure model of ES3 and ES6 including the helical interaction is presented. An in vitro transcribed and folded ES6 sequence differed from that observed in situ, suggesting that chaperones, ribosomal proteins, and/or the tertiary rRNA interaction could be involved in the in vivo folding of ES6. An analysis of the similarities between ES39 structures suggests that it might be under selective constraint to preserve its secondary structure.
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| 2. |
- Moradi, Hossein, 1963-
(författare)
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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
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Simoff, Ivailo, 1971-
(författare)
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Ribosomal proteins L5 and L15 : Functional characterisation of important features, in vivo
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein synthesis is a highly regulated and energy consuming process, during which a large ribonucleoprotein particle called the ribosome, synthesizes new proteins. The eukaryotic ribosome consists of two unequal subunits called: small and large subunits. Both subunits are composed of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). Although rRNAs build the matrix of the ribosome and carries out catalysing of the peptide-bond formation between amino acids, r-proteins also appear to play important structural and functional roles. The primary role of r-proteins is to initiate the correct tertiary fold of rRNA and to organize the overall structure of the ribosome. In this thesis, I focus on two proteins from the large subunit of the eukaryotic ribosome: r-proteins L5 and L15 from bakers yeast S. cerevisiae. Both r-proteins are essential for ribosome function. Their life cycle is primarily associated with rRNA interactions. As a consequence, the proteins show high sequence homology across the species borders. Furthermore, both L5 and L15 are connected to human diseases, which makes the study their role in ribosome biogenesis and ribosome function important. By applying random- and site-directed mutagenesis, coupled with functional complementation tests, I aimed to elucidate functionally regions in both proteins, implicated in transport to the cell nucleus, protein-protein interactions and/or rRNA binding. The importance of individual and multiple amino acid exchanges in the primary sequence of rpL5 and rpL15 were studied in vivo. The obtained results show that S. cerevisiae rpL15 was tolerant to amino acid exchanges in the primary sequence, whereas rpL5 was not. Consequently, A. thaliana rpL15 could substitute for the function of wild type rpL15, whereas none of the tested orthologous proteins to rpL5 could substitute yeast rpL5 in vivo. These observations further emphasize the importance of studying r-proteins as separate entities in the ribosome context.
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