Analysis of translational fidelity in cellular proteins
von Raffaella Garofalo
Datum der mündl. Prüfung:2017-04-03
Erschienen:2019-03-27
Betreuer:Prof. Dr. Marina Rodnina
Gutachter:Prof. Dr. Henning Urlaub
Gutachter:Prof. Dr. Peter Rehling
Zum Verlinken/Zitieren:
http://dx.doi.org/10.53846/goediss-7371
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Name:Dissertation_Raffaella Garofalo_.pdf
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Zusammenfassung
Englisch
The accuracy of protein biosynthesis determines the quality of the proteome and regulates the fitness of the cell. A comprehensive quantification of miscoding events in the cell is essential to understand the basic mechanisms that ensure fidelity as well as the evolutionary consequences of errors of protein synthesis. Impairment in cellular fidelity is associated with numerous diseases and is, due to the increasing number of protein therapeutics, of central biotechnological importance. However, the systematic analysis of miscoding events in vivo is precluded by the limited dynamic range of the available analytical methods. We have developed a method for the quantification of miscoding events such as amino acid misincorporations, which relies on the separation and enrichment of the error-containing peptides from their correct parental ones through multiple rounds of orthogonal chromatography and their absolute quantification by targeted mass spectrometry using isotope-labeled internal standards (AQUA peptides). The dynamic range of the method is linear over more than seven orders of magnitude and it is not restricted to any particular miscoding event, organism, strain or model protein. The analysis of the model protein EF-Tu revealed that near-cognate missense errors are less frequent than expected (median error frequency 10-6) and that the error frequency varies over three orders of magnitude depending on the type of misincorporation and source of EF-Tu. Among the substitutions tested, histidine misincorporations at arginine codons are the most abundant. Furthermore, error frequency varies depending on the amino acid position in EF-Tu by more than one order of magnitude. Positions at which errors are rare are clustered in the EF-Tu/tRNA interface, indicating either an extensive quality control or an higher encoded fidelity of translation. We have also analyzed the effect of aminoglycosides on fidelity and stress response. Most aminoglycosides induce significant miscoding which correlates with the induction of proteins from the the heat-shock stress response and growth defects, underlining the central contribution of miscoding to the bactericidal effect of aminoglycosides. In addition to single-position substitutions, aminoglycosides can induce multiple miscoding events leading to peptides with two or three amino acid substitutions. The frequency of multiple consecutive errors is unexpectedly high and because of the detrimental effects on protein stability, such error clusters may contribute substantially to the bactericidal action of aminoglycosides.
Keywords: ribosomes; targeted mass spectrometry; misincorporations
