%0 Generic
%A Macošek, Jakub
%C Heidelberg
%D 2020
%F heidok:27575
%R 10.11588/heidok.00027575
%T Structural insights into regulation of gene expression
%U https://archiv.ub.uni-heidelberg.de/volltextserver/27575/
%X The thesis comprises two parts investigating structural aspects of various mechanisms  of translational control.  The first chapter investigates a mechanism behind ribosome stalling alleviation.  Poly-proline stretches often induce ribosome stalling, which needs to be  alleviated to translate the mRNA. In bacteria the ribosomes are rescued by Elongation  Factor P (EF-P). EF-P is activated by diverse post-translational modifications  (PTMs) of a positively charged amino acid. Such PTM is the glycosylation of  arginine conserved in approximately 10% of all bacterial species including severe  pathogens (e.g. Pseudomonas aeruginosa). The arginine is glycosylated by a glycosyltransferase  EarP which attaches rhamnose to the arginine. Impairing the  glycosylation reduces the pathogenicity of the bacteria. However, EarP is an uncharacterized  glycosyltranferase as it is only the first documented case of arginine  N-glycosylation in prokaryotes. Hence, the mechanism of EF-P rhamnosylation  by EarP was investigated using nuclear magnetic resonance spectroscopy, X-ray  crystallography and various in vivo and in vitro assays. The atomic structure of  EarP with its substrate dTDP-rhamnose was solved and the in vivo and in vitro  assays together with subsequent studies elucidate the putative mechanism of EarP  rhamnosylation thus providing basis for targeted antibiotic drug design.  The second chapter investigates the translational suppression of the hunchback  (hb) mRNA. The hb mRNA forms during Drosophila development a protein gradient  governing the anterior-posterior body axis formation. The Hunchback protein  gradient results from the suppression of hb mRNA at posterior by a complex of  three proteins – Pumilio (Pum), Nanos and Brain tumor (Brat). Nanos, expressed  in an opposing gradient to Hunchback, provides spatial information. Uniformly  expressed Pum and Brat are RNA binding proteins that specifically recognize the  hb mRNA. The structure of Brat bound to the hb mRNA, and the structure the  complex of Pum and Nanos bound to the hb mRNA have been previously solved.  However, it remains unclear how exactly these three proteins assemble on the hb  mRNA together, and if they are structurally and functional directly linked. The  complex was investigated using modelling based on small-angle X-ray and neutron  scattering data combined with additional restraints from cross-linking/mass  spectrometry. The data were further complemented and validated by various  in vitro assays such as electrophoretic mobility shift assays and isothermal titration  calorimetry. The investigation provides initial insights about the complex and  paves the way for future approaches to fully elucidate the structure of the complex.