Samenvatting
Bacteria use regulatory networks to cope with changing environmental conditions. Studying regulatory mechanisms in bacteria can provide the knowledge needed to develop innovative technologies and applications. Different regulatory mechanisms exist and are coordinated by the cell to react adequately to challenges encountered. One such mechanism is the post-translational phosphorylation of proteins, which can result in the modulation of the protein’s function. A protein subject to post-translational phosphorylation is EF-Tu, the deliverer of aminoacyl-tRNAs to the translating ribosome. EF-Tu is the most abundant protein in many bacteria and is indispensable. Phosphorylation of EF-Tu can be used for the regulation of translation and is involved in the regulation of different biological functions in different bacteria. Phosphorylation of E. coli EF-Tu at Thr382 or B. subtilis EF-Tu at Thr63 inhibits translation. Interestingly, in several studies a total of 31 possible phosphorylation sites have been reported for E. coli EF-Tu in vivo, but all the contributing studies report only a part of all sites. Moreover, quantitative information and information about the dynamics of phosphorylation during growth and under stress conditions is missing. The physiological role of most sites also remains to be elucidated.To understand regulation of translation in bacteria, the phosphorylation of EF-Tu was studied in E. coli. An expression system was designed to express His-tagged EF-Tu under physiological relevant conditions. This system enables the purification of EF-Tu and the study of the phosphorylation states of EF-Tu in different growth phases and under different stress conditions. The phosphorylation state of EF-Tu was determined by mass spectroscopy. Phosphorylation is gedetecteerd at two new sites, Ser1 and Tyr69, at low levels. Localisation of Ser1 on the EF-Tu structure indicates that phosphorylation at this site might influence tRNA binding. The low phosphorylation levels observed in this study and a comparison with other studies that describe EF-Tu phosphorylation suggests the occurrence of non-functional ‘background’ phosphorylation of EF-Tu.
In addition, point mutations were made in the tagged EF-Tu to study the role of specific phosphorylation sites by analysing how these mutations influence growth of E. coli and the phosphorylation pattern of EF-Tu. Mutations at Tyr331 strongly affect growth, an indication that phosphorylation at this site might have an impact on EF-Tu functioning. An in silico analysis provided the first insights into the effect of phosphorylation of Tyr331 on EF-Tu functioning. Possibly, tRNA binding is affected upon phosphorylation of Tyr331.
Together, these results provide a starting point for a larger study on EF-Tu phosphorylation that aims to unravel the role of post-translational modifications of EF-Tu in bacterial stress response and persistence.
| Datum prijs | jul. 2019 |
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| Originele taal | English |
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