Véronique Arluison

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA–RNA interactions.

The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators

In Escherichia coli, the essential motor protein Rho promotes transcription termination in a tightly controlled manner that is not fully understood. Here, we show that the general post‐transcriptional regulatory protein Hfq associates with Rho to regulate Rho function. The Hfq:Rho complex can be further stabilized by RNA bridging both factors in a configuration that inhibits the ATP hydrolysis and duplex unwinding activities of Rho and that mediates transcription antitermination at Rho‐dependent terminators in vitro and in vivo. Antitermination at a prototypical terminator (λtR1) requires Hfq binding to an A/U‐rich transcript region directly upstream from the terminator. Antitermination is modulated by trans‐acting factors (NusG or nucleic acid competitors) that affect Hfq association with Rho or RNA. These data unveil a new Hfq function and a novel transcription regulatory mechanism with potentially important implications for bacterial RNA metabolism, gene silencing, and pathogenicity.

Sm-like protein Hfq: Location of the ATP-binding site and the effect of ATP on Hfq–RNA complexes

Sm‐like proteins are ubiquitous ring‐shaped oligomers that exhibit a variety of nucleic acid‐binding activities. They have been linked functionally to various cellular events involving RNA, and it is generally believed that their activity is exerted via the passive binding of nucleic acids. Our earlier studies of the Sm‐like Escherichia coli protein Hfq provided the first evidence indicating that Hfq is an ATP‐binding protein. Using a combination of biochemical and genetic techniques, we have now determined a plausible ATP‐binding site in Hfq and tested Hfqs ATP‐binding affinity and stoichiometry. The results of RNA footprinting and binding analyses suggest that ATP binding by the Hfq–RNA complex results in its significant destabilization. RNA footprinting indicates deprotection of Hfq‐bound RNA tracts in the presence of ATP, suggestive of their release by the protein. The results reported herein broaden the scope of potential in vivo roles for Hfq and other Sm‐like proteins.

Auto-assembly of E. coli DsrA small noncoding RNA: Molecular characteristics and functional consequences

RNA molecules are important factors involved in different cellular processes and have a multitude of roles in the cell. These roles include serving as a temporary copy of genes used for protein synthesis or functions in translational machinery. Interestingly, RNA is so far the only biological molecule that serves both as a catalyst (like proteins) and as information storage (like DNA). However, in contrast to proteins well known to be able to self-associate in order to maintain the architecture of the cell, such RNA polymers are not prevalent in cells and are usually not favored by the flexibility of this molecule. In this work, we present evidence that such a polymer of a natural RNA, the DsrA RNA, exists in the bacterial cell. DsrA is a small noncoding RNA (87 nucleotides) of Escherichia coli that acts by base-pairing to mRNA in order to control the translation and the turnover of some mRNA, including rpoS mRNA, which encodes the σs RNA polymerase subunit involved in bacterial stress response. A putative model is proposed for the structure of this RNA polymer. Although the function of this polymerization is not known completely, we propose that the formation of such a structure could be involved in the regulation of DsrA ncRNA concentration in vivo or in a quality control mechanism used by the cell to eliminate misfolded RNAs.

Involvement of HFq protein in the post-transcriptional regulation of E. coli bacterial cytoskeleton and cell division proteins

Keywords: Cell Division Reference EPFL-ARTICLE-181924 Record created on 2012-10-26, modified on 2017-05-12

Auto-assembly as a new regulatory mechanism of noncoding RNA

An abstract is not available for this article.

Thermodynamics of the β2 association in light‐harvesting complex I of Rhodospirillum rubrum

The core light‐harvesting LH1 protein from Rhodospirillum rubrum can dissociate reversibly in the presence of n‐octyl‐β‐d‐glucopyranoside into smaller subunit forms, exhibiting a dramatic blue‐shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820u2003nm (B820) and a monomer absorbing at 777u2003nm (B777). In the presence of n‐octyl‐β‐D‐glucopyranoside, we have previously shown that the B820 form is not only constituted by the αβ heterodimer alone, but that it exists in an equilibrium between the αβ heterodimer and β2 homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoritical model for αβ and β2, we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non‐covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes.

Dynamic Competition for the Proximal RNA Binding Site of Hfq Drives Efficient Annealing of Small Noncoding RNA and mRNA

Hfq, a bacterial RNA chaperone, stimulates specific RNA-RNA interactions by inducing RNA unwinding or RNA-RNA annealing. However, it is still unclear how Hfq can selectively achieve two completely opposite functions--annealing, and unwinding--depending on RNA substrates. Also, basic properties like an oligomeric state of an active form is under debate. Here we describe single-molecule fluorescence studies on Hfq-mediated annealing of DsrA, a small noncoding regulatory RNA of E. coli, and its mRNA target rpoS, which encodes the σStranscription factor. Our results reveal that a single hexamer of Hfq stimulates DsrA-rpoS annealing by simultaneously binding both RNAs using same RNA binding site on the proximal surface of Hfq. The competition for the same binding site of the two RNAs makes the RNA-Hfq interaction dynamic, but drives more efficient annealing. When the Hfq-binding sequence exists only in one of the two RNAs, RNA unwinding can occur due to the reduced stability of the RNA caused by the partial unwinding of the RNA upon Hfq binding on the specific binding site.