RICERCA ITALIANA     

DNA-binding properties of the bacterial nucleoid protein H-NS: molecular analysis focused on DNA recognition at the nucleation points.

Università degli Studi di Camerino

Abstract

DNA-packaging and structural organization of chromatin in bacterial cells relies on a heterogeneous group of DNA-binding proteins (H-NS, HU, IHF, StpA, Lrp, FIS and Dps) collectively named histone-like proteins.
H-NS is one of the most abundant nucleoid-associated proteins in Escherichia coli (as well as in other Enterobacteriaceae). In addition to participating in the structural organization of bacterial chromosome, it is also involved in the regulation, primarily at the transcriptional level, of a select and fairly large number of genes. The structural basis for H-NS function seems to reside in the capacity of this protein to recognize and bind specifically to intrinsically curved regions of DNA, that are frequently found at promoter regions. From past work we know that these regions very often contain adenine-tract sequences, and function as upstream recognition elements; however the role played by these curvatures is almost unknown. In this context, several crucial questions remain unanswered, relating to a) the dynamic procedure by which H-NS discriminates between specific and non-specific DNA binding, b) conformational changes of the protein upon DNA binding, possibly involving rearrangements of adjacent domains within a single monomer and also different quaternary structures, c) the inherent structure and flexibility of the DNA sequences representing a preferential target for H-NS, d) the different degree of DNA deformation upon H-NS binding.
The aim >>>

Principal Investigator

Roberto SPURIO Università degli Studi di CAMERINO

Research Objectives

The main objectives and highlights in this research project are summarized below:
1. identification of a "consensus" sequence recognized by E.coli H-NS, by means of footprinting experiments.
We plan to use the DNA-binding C-terminal domain of H-NS (H-NS 1-88) to extend a preliminary search (from which we have obtained some preliminary data) of preferential binding to selected sites within the promoter regions of genes known to be regulated by H-NS.
A similar analysis will be undertaken using the full-length protein and high resolution footprinting experiments, coupled with the use of fast kinetic instrument.
This part of the project will be carried out at the University of Camerino, with partial involvement of a PhD student and with a PhD fellow supported by this grant working full-time.
2. structural analysis of the molecular basis of the specificity governing H-NS-DNA interaction by multidimensional heteronuclear NMR spectroscopy.
This task will be tackled by Dr. Marco Sette (University of Rome), who has the scientific background necessary to approach all different aspects of the structural studies described in this program.
In addition to some instruments available at the University of Rome, he can count on machine-time available on a Bruker AVANCE 600 MHz NMR instrument equipped with cryoprobe, located at IRBM (Pomezia, Rome), and an 800 MHz VARIAN, located at the National Institute for Medical Research >>>

Timescale

24 months

National and international background

The bacterial nucleoid is distinguished from the eukaryotic nucleus in a number of fundamental ways, not the least of which are the lack of a membrane separating the chromosomal DNA from the cytosol and the lack of a stable and ordered nucleosomal structure to the DNA. In the absence of histones, a set of abundant "nucleoid associated" proteins coats a significant fraction of the bacterial chromosome. Earlier reviews discussing these proteins can be found in Drlica and Rouvier-Yaniv (1987), Schmid (1988), Murphy and Zimmermann (1997). Organization of the condensed chromosome in the nucleoid involves the formation of a set (40-200) of topologically constrained DNA loops per chromosome ranging from 10 to 100 Kbp in size. The DNA within the topologically isolated domains is negatively supercoiled, and half of the linking number deficit is believed to be primarily stabilized by interactions with the major nucleoid proteins, probably in conjunction with polyamines (e.g. spermidine). The current view is that the E.coli nucleoid is dominated by five different proteins: H-NS and its paralogue StpA, HU and its paralogue IHF and Fis. Assuming three chromosome equivalents per exponentially growing cell in rich media, along with their DNA binding site sizes and cellular levels, one can estimate that about 20% of the chromosome is bound by these proteins.
1- General features of H-NS
H-NS (histone-like nucleoid-structuring protein), the subject of this research program, is >>>