Identification of in-vivo formed sRNA-protein complexes

In recent years, quite a number of small, non-coding RNAs (sRNAs) have been identified in bacteria, but only a few have been functionally characterised. An abundant class are trans-encoded sRNAs. Yet little is known about the detailed mechanisms of action for those regulatory RNAs, especially in the case of multiple mRNA targeting. The protein Hfq is supposed to play an important role in the action of the three trans-encoded sRNAs RybB, GcvB and InvR in Salmonella. However, several other proteins might also be involved in target regulation in vivo. To get deeper insight in those regulatory mechanisms, the goal of the project is the isolation, identification and characterisation of proteins that bind in vivo to the sRNA, the target mRNA or the complex of both. For the specific and efficient isolation of in vivo formed complexes, an affinity purification strategy will be used. Therefore a suitable plasmid vector will be constructed, that allows the fusion of an RNA aptamer sequence to the 5` or the 3` end of the sRNA or the mRNA target gene. Following in vivo expression and assembly of the RNAs and the proteins in Salmonella, the putative complexes will be isolated by affinity chromatography, for which different binding and elution strategies can be used. The identification of the proteins will then be realised by mass spectrometry. Depending on the nature of the identified proteins, different in vitro and in vivo experiments will be performed to characterise the interaction between RNA and protein. All these different steps will be implemented with the final goal to better understand the regulation mechanisms mediated by sRNAs.