Cellular Electron Microscopy Imaging Reveals the Localization of the Hfq Protein Close to the Bacterial Membrane

Background: Hfq is a bacterial protein involved in several aspects of nucleic acid transactions, but one of its bestcharacterized functions is to affect the post-transcriptional regulation of mRNA by virtue of its interactions with stressrelated small regulatory (sRNA). Methodology and Principal Finding: By using cellular imaging based on the metallothionein clonable tag for electron microscopy, we demonstrate here that in addition to its localization in the cytoplasm and in the nucleoid, a significant amount of Hfq protein is located at the cell periphery. Simultaneous immunogold detection of specific markers strongly suggests that peripheral Hfq is close to the bacterial membrane. Because sRNAs regulate the synthesis of several membrane proteins, our result implies that the sRNA- and Hfq-dependent translational regulation of these proteins takes place in the cytoplasmic region underlying the membrane. Conclusions: This finding supports the proposal that RNA processing and translational machineries dedicated to membrane protein translation may often be located in close proximity to the membrane of the bacterial cell

Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing

Hfq is a key regulator involved in multiple aspects of stress tolerance and virulence of bacteria. There has been an intriguing question as to how this RNA chaperone achieves two completely opposite functions—annealing and unwinding—for different RNA substrates. To address this question, we studied the Hfq-mediated interaction of fragments of a non-coding RNA, DsrA, with its mRNA target rpoS by using single-molecule fluorescence techniques. These experiments permitted us to observe the mechanistic steps of Hfq-mediated RNA annealing/unwinding at the single-molecule level, for the first time. Our real-time observations reveal that, even if the ring-shaped Hfq displays multiple binding sites for its interaction with RNA, the regulatory RNA and the mRNA compete for the same binding site. The competition makes the RNA-Hfq interaction dynamic and, surprisingly, increases the overall annealing efficiency by properly aligning the two RNAs. We furthermore reveal that when Hfq specifically binds to only one of the two RNAs, the unwinding process dominates over the annealing process, thus shedding a new light on the substrate selectivity for annealing or unwinding. Finally, our results demonstrate for the first time that a single Hfq hexamer is sufficient to facilitate sRNA–mRNA annealing

Biophysical Methods for the Elucidation of the S-Layer Proteins/Metal Interaction

Surface-layers (S-layers) are macromolecular paracrystalline arrays of proteins or glycoproteins that can self-assemble into 2-dimensional semi-permeable meshworks to overlay the cell surface of many bacteria and archaea. They usually assemble into lattices with oblique, square or hexagonal symmetry and serve as an interface between the bacterial cell and the environment. Isolated S-layers can recrystallize into two-dimensional regular arrays in suspension or on various surfaces, thus being an appropriate material for several bionanotechnological purposes. Promising applications of S-layers include their use as biotemplates for the capture of metal ions or the synthesis of metal nanoclusters. Considering the use of S-layers as biotemplates for the organization of metal ions or metallic nanoclusters, research on potential of surface layer proteins (SLP) and metals can be understood as an interdisciplinary field, in which different biophysical techniques supply complementary information. In this review, we discuss the SLP as native or engineered “bottom-up” building blocks for metal immobilization structures. We also describe the biophysical techniques used to analyze metal binding properties as well as the information obtained from the investigation of these structures.Centro de Investigación y Desarrollo en Criotecnología de AlimentosFacultad de Ciencias Exacta

Techniques to Analyze sRNA Protein Cofactor Self-Assembly In Vitro

Post-transcriptional control of gene expression by small regulatory noncoding RNA (sRNA) needs protein accomplices to occur. Past research mainly focused on the RNA chaperone Hfq as cofactor. Nevertheless, recent studies indicated that other proteins might be involved in sRNA-based regulations. As some of these proteins have been shown to self-assemble, we describe in this chapter protocols to analyze the nano-assemblies formed. Precisely, we focus our analysis on Escherichia coli Hfq as a model, but the protocols presented here can be applied to analyze any polymer of proteins. This chapter thus provides a guideline to develop commonly used approaches to detect prokaryotic protein self-assembly, with a special focus on the detection of amyloidogenic polymers

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

Twins, quadruplexes, and more: functional aspects of native and engineered RNA self-assembly in vivo

The primacy and power of RNA in governing many processes of life has begun to be more fully appreciated in both the discovery and inventive sciences. A variety of RNA interactions regulate gene expression, and structural self-assembly underlies many of these processes. The understanding sparked by these discoveries has inspired and informed the engineering of novel RNA structures, control elements, and genetic circuits in cells. Many of these engineered systems are built up fundamentally from RNA–RNA interactions, often combining modular, rational design with functional selection and screening. It is therefore useful to review the particular class of RNA-based regulatory mechanisms that rely on RNA self-assembly either through homomeric (self–self) or heteromeric (self–nonself) RNA–RNA interactions. Structures and sequence elements within individual RNAs create a basis for the pairing interactions, and in some instances can even lead to the formation of RNA polymers. Example systems of dimers, multimers, and polymers are reviewed in this article in the context of natural systems, wherein the function and impact of self-assemblies are understood. Following this, a brief overview is presented of specific engineered RNA self-assembly systems implemented in vivo, with lessons learned from both discovery and engineering approaches to RNA–RNA self-assembly

Thermodynamic aspects of the self-assembly of DsrA, a small noncoding RNA from Escherichia coli

DsrA is an Escherichia coli small noncoding RNA that acts by base pairing to some mRNAs in order to control their translation and turnover. It was recently shown that DsrA is able to self-associate in a way similar to DNA and to build nanostructures. Although functional consequence of this RNA self-assembly in vivo is not yet understood, the formation of such an assemblage more than likely influences the noncoding RNA function. We report here for the first time the thermodynamic basis of this natural RNA self-assembly. In particular we show that assembling of the ribonucleic acid is enthalpy driven and that the versatility of the RNA molecule is important for the polymerisation; indeed, an equivalent DNA sequence is unable to make a nanoassembly. The origin of the difference is discussed herein