Cryo soft X-ray tomography to explore Escherichia coli nucleoid remodeling by Hfq master regulator.

The bacterial chromosomic DNA is packed within a membrane-less structure, the nucleoid, due to the association of DNA with proteins called Nucleoid Associated Proteins (NAPs). Among these NAPs, Hfq is one of the most intriguing as it plays both direct and indirect roles on DNA structure. Indeed, Hfq is best known to mediate post-transcriptional regulation by using small noncoding RNA (sRNA). Although Hfq presence in the nucleoid has been demonstrated for years, its precise role is still unclear. Recently, it has been shown in vitro that Hfq forms amyloid-like structures through its C-terminal region, hence belonging to the bridging family of NAPs. Here, using cryo soft X-ray tomography imaging of native unlabeled cells and using a semi-automatic analysis and segmentation procedure, we show that Hfq significantly remodels the Escherichia coli nucleoid. More specifically, Hfq influences nucleoid density especially during the stationary growth phase when it is more abundant. Our results indicate that Hfq could regulate nucleoid compaction directly via its interaction with DNA, but also at the post-transcriptional level via its interaction with RNAs. Taken together, our findings reveal a new role for this protein in nucleoid remodeling in vivo, that may serve in response to stress conditions and in adapting to changing environments.

Application of Synchrotron Radiation Circular Dichroism for RNA Structural Analysis.

Circular dichroism (CD) spectroscopy is a fast and simple technique providing important information about the conformation of nucleic acids, proteins, sugars, lipids, and their interactions between each other. This electronic absorption spectroscopy method is extremely sensitive to any change in molecular structure containing asymmetric molecules. While numerous reviews describe how to analyze deoxyribonucleic acid (DNA) structures using CD, analyses of ribonucleic acids (RNAs) are scarce. Nevertheless, RNAs are important molecules involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) extending the spectral range down to 170 nm, improving structural analysis of RNA, including the analysis of helical parameters and alternative structures found in RNA. The effects of temperature to measure thermodynamic parameters and analyze ribonucleoprotein complexes will also be presented.

The Bacterial Amyloid-Like Hfq Promotes In Vitro DNA Alignment.

The Hfq protein is reported to be involved in environmental adaptation and virulence of several bacteria. In Gram-negative bacteria, Hfq mediates the interaction between regulatory noncoding RNAs and their target mRNAs. Besides these RNA-related functions, Hfq is also associated with DNA and is a part of the bacterial chromatin. Its precise role in DNA structuration is, however, unclear and whether Hfq plays a direct role in DNA-related processes such as replication or recombination is controversial. In previous works, we showed that Escherichia coli Hfq, or more precisely its amyloid-like C-terminal region (CTR), induces DNA compaction into a condensed form. In this paper, we evidence a new property for Hfq; precisely we show that its CTR influences double helix structure and base tilting, resulting in a strong local alignment of nucleoprotein Hfq:DNA fibers. The significance of this alignment is discussed in terms of chromatin structuration and possible functional consequences on evolutionary processes and adaptation to environment.