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Elongation factor 2: A key component of the translation machinery in eukaryotes: Properties of yeast elongation factor 2 studied in vivo
Södertörn University, School of Life Sciences. Stockholms universitet, Wenner-Grens institut.
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-Gren Institute for Experimental Biology, Stockholm university , 2008. , 56 p.
Keyword [en]
Elongation factor 2, yeast, ribosome, phosphorylation, Switch I, site-directed mutagenesis, functional complementation
National Category
Cell Biology
Identifiers
URN: urn:nbn:se:sh:diva-32038ISBN: 978-91-7155-634-9 (print)OAI: oai:DiVA.org:sh-32038DiVA: diva2:1073753
Public defence
2008-06-04, MA331, Alfred Nobels allé 7, Huddinge, 13:00 (English)
Opponent
Supervisors
Available from: 2017-02-13 Created: 2017-02-13 Last updated: 2017-02-13Bibliographically approved
List of papers
1. Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast
Open this publication in new window or tab >>Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast
2007 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 274, no 20, 5285-5297 p.Article in journal (Refereed) Published
Abstract [en]

Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.

National Category
Cell Biology
Identifiers
urn:nbn:se:sh:diva-14205 (URN)10.1111/j.1742-4658.2007.06054.x (DOI)000249882400009 ()17892487 (PubMedID)2-s2.0-34848907218 (Scopus ID)
Available from: 2011-12-20 Created: 2011-12-19 Last updated: 2017-02-13Bibliographically approved
2. Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae
Open this publication in new window or tab >>Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae
2008 (English)In: Biochimie, ISSN 0300-9084, E-ISSN 1638-6183, Vol. 90, no 5, 736-748 p.Article in journal (Refereed) Published
Abstract [en]

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, 169M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, 169M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and 169M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to 169) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:sh:diva-14152 (URN)10.1016/j.biochi.2008.01.006 (DOI)000256256000007 ()18267126 (PubMedID)2-s2.0-43049110270 (Scopus ID)
Available from: 2011-12-18 Created: 2011-12-16 Last updated: 2017-02-13Bibliographically approved
3. The functional importance of the N- and C-terminal regions in elongation factor 2 from S. cerevisiae
Open this publication in new window or tab >>The functional importance of the N- and C-terminal regions in elongation factor 2 from S. cerevisiae
(English)Manuscript (preprint) (Other academic)
National Category
Biological Sciences
Identifiers
urn:nbn:se:sh:diva-32039 (URN)
Note

Som manuskript i avhandling. As manuscript in dissertation.

Available from: 2008-05-13 Created: 2017-02-13 Last updated: 2017-02-13Bibliographically approved
4. Functional features of the C-terminal region of yeast ribosomal protein L5
Open this publication in new window or tab >>Functional features of the C-terminal region of yeast ribosomal protein L5
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 alpha-helix with a positively charged surface that is involved in protein-5S rRNA interaction. Formation of an YrpL5 center dot 5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the alpha-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 alpha-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 alpha-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.

National Category
Biochemistry and Molecular Biology Genetics
Identifiers
urn:nbn:se:sh:diva-14121 (URN)10.1007/s00438-008-0369-7 (DOI)000258902300007 ()18751732 (PubMedID)2-s2.0-51349101530 (Scopus ID)
Available from: 2011-12-18 Created: 2011-12-16 Last updated: 2017-02-13Bibliographically approved

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