Elongation factor 2: A key component of the translation machinery in eukaryotes : Properties of yeast elongation factor 2 studied in vivo
Sammanfattning: 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.
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