For many years H-NS has been considered as a DNA-binding protein without a sequence specificity, having a role of nucleoid structuring protein with a global effect on gene expression. The capacity of H-NS to repress a wide range of genes was attributed to its characteristic of recognizing and preferentially binding AT-reach sequences, and intrinsically curved DNA. Recently, quantitative analyses of the H-NS binding sites at the E. coli proU promoter, has demonstrated that H-NS recognizes a 10bp sequence with a high affinity. Moreover, comparing footprints results obtained by the binding of the isolated C-terminal domain of H-NS (responsible for the binding capacity) on five different target promoters (Stella S. Ph.D Thesis, 2005), and combining footprinting data from different sources, an H-NS binding motif has been found. The derived motif was used to predict potential binding sites, and these predictions were confirmed by following footprinting analyses. However the existence of specific binding sites for H-NS, only partially explains the silencing effect of H-NS on different promoters. A proposed model, to explain the mechanism by which H-NS silences extended regions of DNA, suggests that the protein nucleate at high-affinity sites located in the core promoter of target genes, and subsequently it spreads along secondary sites of lower affinity. In this way the protein occupies the promoter region and allows the formation of nucleoprotein complexes of higher order structure. In this project we wanted to analyze the fundamental steps of H-NS/DNA interactions from a dynamical point of view, to understand the molecular mechanisms leading the oligomerization of H-NS along its target DNA, in a temporal resolution. For this purpose we set up a series of quantitative time-resolved DNaseI footprinting assays. We used a KinTek quenched-flow apparatus, that allows the analysis of the association of a protein on its target, at increasing times of protein/DNA interaction. Since H-NS protein directly affects the expression of its own gene, as target DNA we used the natural hns promoter from E. coli. We also investigated the kinetics of H-NS binding upon a DNA fragment, where the isolated H-NS binding motif was introduced into a portion of the Tetracycline Resistance gene, for which H-NS has a general low affinity. Moreover we compared time-resolved data with equilibrium footprinting assays carried out using the isolated C-terminal domain, responsible for the DNA binding.
|Titolo:||Oligomerization Dynamics of the E. coli Nucleoid Protein H-NS on its target DNA|
|Data di pubblicazione:||2008|
|Appare nelle tipologie:||Tesi di dottorato (Pregresso)|