"In bacteria, DNA transposons play a key role in transferring antibiotic resistance, which poses a major threat to human health. I want to understand how this transfer can be controlled. One of the regulatory pathways applied in vivo uses specific small RNAs (sRNAs) controlling translation of the transposase mRNA. This is achieved using the general bacterial RNA regulatory machinery, and is mediated by the small RNA chaperone, HFQ. HFQ is a highly conserved, SM-like protein that modifies the secondary and tertiary structure of sRNAs and mRNAs, supports RNA unfolding and annealing and thereby regulates mRNA translation. In addition, HFQ also recruits the RNA degradation machinery through interaction with the endonuclease RNaseE and the poly(A)-polymerase PAP-I. Now I aim to understand the mechanism of sRNA-mediated regulation of transposition and the general gene silencing process in bacteria, applying an integrated structural biology approach. Using fluorescence anisotropy, SPR, FRET, NMR spectroscopy and X-ray crystallography, I will study the structural changes of Tn10 derived RNAs upon binding to HFQ, the structure of the ribonucleoprotein complexes involved and the dynamics of RNA-folding and annealing. Furthermore, to investigate the intimate link between sRNA based translation regulation and RNA degradation, I will also analyze how HFQ interacts with RNaseE and PAP-I, and how these interactions affect the function of these RNA processing factors.
My study will not only reveal the mechanism of sRNA based regulation of transposition, but also provide insights into the general RNA chaperoning functions of HFQ and a comprehensive picture about the macromolecular complexes and dynamic events involved in sRNA mediated gene regulation. On the long term, I envision that down regulation of transposition may offer a way to inhibit the transmission of antibiotic resistances, which would be of greatest medical interest."
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