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Functional features of the C-terminal region of yeast ribosomal protein L5
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
Stockholm University, Faculty of Science, The Wenner-Gren Institute .
2008 (English)In: Molecular Genetics and Genomics, ISSN 1617-4615, E-ISSN 1617-4623, Vol. 280, no 4, 337-350 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to analyze the functional importance of the C-terminus of the essential yeast ribosomal protein L5 (YrpL5). Previous studies have indicated that the C-terminal region of YrpL5 forms an α-helix with a positively charged surface that is involved in protein–5S rRNA interaction. Formation of an YrpL5·5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the α-helix and the positively charged surface for YrpL5 function in Saccharomyces cerevisiae using site directed mutagenesis in combination with functional complementation. Alterations in the sequence forming the putative α-helix affected the functional capacity of YrpL5. However, the effect did not correlate with a decreased ability of the protein to bind to 5S rRNA as all rpL5 mutants tested were imported to the nucleus whether or not the α-helix or the positively charged surface were intact. The alterations introduced in the C-terminal sequence affected the growth rate of cells expressing mutant but functional forms of YrpL5. The reduced growth rate was correlated with a reduced ribosomal content per cell indicating that the alterations introduced in the C-terminus interfered with ribosome assembly.

Place, publisher, year, edition, pages
2008. Vol. 280, no 4, 337-350 p.
Keyword [en]
Functional complementation, Mutation analysis, Ribosomal protein L5, S. cerevisiae
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
URN: urn:nbn:se:su:diva-25114DOI: 10.1007/s00438-008-0369-7ISI: 000258902300007OAI: oai:DiVA.org:su-25114DiVA: diva2:198931
Available from: 2008-05-13 Created: 2008-05-12 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Elongation factor 2: A key component of the translation machinery in eukaryotes: Properties of yeast elongation factor 2 studied in vivo
Open this publication in new window or tab >>Elongation factor 2: A key component of the translation machinery in eukaryotes: Properties of yeast elongation factor 2 studied in vivo
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Synthesis of proteins is performed by the ribosome, a large ribonucleoprotein complex. Apart from the ribosome, numerous protein factors participate in this process. Elongation factor 2 (eEF2) is one of these factors. eEF2 is an essential protein with a mol. mass of about 100 kDa. The amino acid sequence of eEF2 is highly conserved in different organisms. eEF2 from S. cerevisiae contains 842 amino acids. The role of eEF2 in protein synthesis is to participate in the translocation of tRNAs from the A- and P-sites on the ribosome to the P- and E-sites. This movement of tRNAs is accompanied by a simultaneous movement of mRNA by one codon. eEF2 consists of six domains referred to as domains G, G′ and II-V, belongs to the G-protein super-family and possesses all structural motifs characterizing proteins in this family. eEF2 binds to the ribosome in complex with GTP. After GTP hydrolysis and translocation, it leaves the ribosome bound to GDP. The rate of protein synthesis in the cell can be regulated by phosphorylation of eEF2. Phosphorylation occurs on two threonine residues, situated in the G domain of the factor. Phosphorylation of eEF2 is catalysed by Rck2-kinase in yeast which is activated in response to osmotic stress. Despite the high degree of conservation of the threonine residues, they are not essential for yeast cell under normal growth conditions. However, under mild osmotic stress the growth rate of the cells lacking threonine residues was decreased. Region where threonine residues are located, called Switch I. Cryo-EM reconstruction shows that this region adopts ordered conformation when the eEF2•GTP complex is bound to the ribosome but became structurally disordered upon GTP hydrolysis. Mutagenesis of individual amino acids in Switch I resulted in both functional and non-functional eEF2 depending on the site of mutation and the substituting amino acid. Both functional and non-functional Switch I mutants were able to bind to the ribosome, indicating that mutations did not abolish the capacity of the factor to bind GTP. Yeast eEF2 with Switch I region from E. coli was able to substitute the wild type protein in vivo, though the growth rate of these cells was severely impaired. The eEF2-dependent GTP hydrolysis can be activated by ribosome from heterologous sources as seen in vitro. However, eEF2 from A. thaliana, D. melanogaster and S. solfataricus could not substi-tute yeast eEF2 in vivo. This may indicate additional roles of eEF2 in the yeast cell, apart from translocation itself.

Place, publisher, year, edition, pages
Stockholm: Wenner-Grens institut för experimentell biologi, 2008. 56 p.
Keyword
Elongation factor 2, yeast, ribosome, phosphorylation, Switch I, site-directed mutagenesis, functional complementation
National Category
Cell Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-7733 (URN)978-91-7155-634-9 (ISBN)
Public defence
2008-06-04, MA331, Söderstörns högskola, Alfred Nobels allé 7, Huddinge, 13:00
Opponent
Supervisors
Available from: 2008-05-13 Created: 2008-05-12Bibliographically approved
2. Ribosomal proteins L5 and L15: Functional characterisation of important features, in vivo
Open this publication in new window or tab >>Ribosomal proteins L5 and L15: Functional characterisation of important features, in vivo
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Green Institute, Stockholm University, 2009. 50 p.
National Category
Biochemistry and Molecular Biology
Research subject
Cellbiology
Identifiers
urn:nbn:se:su:diva-27731 (URN)978-91-7155-896-1 (ISBN)
Public defence
2009-06-16, MB 503, Södertörns högskola, Alfred Nobels allé 7, Huddinge, 10:00 (English)
Opponent
Supervisors
Available from: 2009-05-25 Created: 2009-05-14 Last updated: 2009-05-15Bibliographically approved
3. 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
Open this publication in new window or tab >>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 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: The Wenner-Gren Institute, Stockholm University, 2011. 58 p.
Keyword
Ribosome, Ribosomal protein, Elongation factor, Translation
National Category
Cell Biology
Research subject
Cell Biology
Identifiers
urn:nbn:se:su:diva-63520 (URN)978-91-7447-378-0 (ISBN)
Public defence
2011-11-25, lecture room E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20 C, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2011-11-02 Created: 2011-10-21 Last updated: 2011-10-26Bibliographically approved

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