RNA Nanostructure Molecular Imaging.

Atomic force and transmission electron microscopies (AFM/TEM) are powerful tools to analyze RNA-based nanostructures. While cryo-TEM analysis allows the determination of near-atomic resolution structures of large RNA complexes, this chapter intends to present how RNA nanostructures can be analyzed at room temperature on surfaces. Indeed, TEM and AFM analyses permit the conformation of a large population of individual molecular structures to be observed, providing a statistical basis for the variability of these nanostructures within the population. Nevertheless, if double-stranded DNA molecular imaging has been described extensively, only a few investigations of single-stranded DNA and RNA filaments have been conducted so far. Indeed, technique for spreading and adsorption of ss-molecules on AFM surfaces or TEM grids is a crucial step to avoid disturbing RNA conformation on the surface. In this chapter, we present a specific method to analyze RNA assemblies and RNA-protein complexes for molecular microscopies.

Multiple approaches for the investigation of bacterial small regulatory RNAs self-assembly.

RNAs are flexible molecules involved in a multitude of roles in the cell. Specifically, noncoding RNAs (i.e., RNAs that do not encode a protein) have important functions in the regulation of biological processes such as RNA decay, translation, or protein translocation. In bacteria, most of those noncoding RNAs have been shown to be critical for posttranscriptional control through their binding to the untranslated regions of target mRNAs. Recent evidence shows that some of these noncoding RNAs have the propensity to self-assemble in prokaryotes. Although the function of this self-assembly is not known and may vary from one RNA to another, it offers new insights into riboregulation pathways. We present here the various approaches that can be used for the detection and analysis of bacterial small noncoding RNA self-assemblies.

Effects of Hfq on the conformation and compaction of DNA.

Hfq is a bacterial pleiotropic regulator that mediates several aspects of nucleic acids metabolism. The protein notably influences translation and turnover of cellular RNAs. Although most previous contributions concentrated on Hfq's interaction with RNA, its association to DNA has also been observed in vitro and in vivo. Here, we focus on DNA-compacting properties of Hfq. Various experimental technologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy and small angle neutron scattering have been used to follow the assembly of Hfq on DNA. Our results show that Hfq forms a nucleoprotein complex, changes the mechanical properties of the double helix and compacts DNA into a condensed form. We propose a compaction mechanism based on protein-mediated bridging of DNA segments. The propensity for bridging is presumably related to multi-arm functionality of the Hfq hexamer, resulting from binding of the C-terminal domains to the duplex. Results are discussed in regard to previous results obtained for H-NS, with important implications for protein binding related gene regulation.

The polymorphisms of the chromatin fiber.

In eukaryotes, the genome is packed into chromosomes, each consisting of large polymeric fibers made of DNA bound with proteins (mainly histones) and RNA molecules. The nature and precise 3D organization of this fiber has been a matter of intense speculations and debates. In the emerging picture, the local chromatin state plays a critical role in all fundamental DNA transactions, such as transcriptional control, DNA replication or repair. However, the molecular and structural mechanisms involved remain elusive. The purpose of this review is to give an overview of the tremendous efforts that have been made for almost 40 years to build physiologically relevant models of chromatin structure. The motivation behind building such models was to shift our representation and understanding of DNA transactions from a too simplistic 'naked DNA' view to a more realistic 'coated DNA' view, as a step towards a better framework in which to interpret mechanistically the control of genetic expression and other DNA metabolic processes. The field has evolved from a speculative point of view towards in vitro biochemistry and in silico modeling, but is still longing for experimental in vivo validations of the proposed structures or even proof of concept experiments demonstrating a clear role of a given structure in a metabolic transaction. The mere existence of a chromatin fiber as a relevant biological entity in vivo has been put into serious questioning. Current research is suggesting a possible reconciliation between theoretical studies and experiments, pointing towards a view where the polymorphic and dynamic nature of the chromatin fiber is essential to support its function in genome metabolism.

Pack, unpack, bend, twist, pull, push: the physical side of gene expression.

Molecular motors such as polymerases produce physical constraints on DNA and chromatin. Recent techniques, in particular single-molecule micromanipulation, provide estimation of the forces and torques at stake. These biophysical approaches have improved our understanding of chromatin behaviour under physiological physical constraints and should, in conjunction with genome wide and in vivo studies, help to build more realistic mechanistic models of transcription in the context of chromatin. Here, we wish to provide a brief overview of our current knowledge in the field, and emphasize at the same time the importance of DNA supercoiling as a major parameter in gene regulation.

Chromatin structure and radiation-induced DNA damage: from structural biology to radiobiology.

Genomic DNA in eukaryotic cells is basically divided into chromosomes, each consisting of a single huge nucleosomal fiber. It is now clear that chromatin structure and dynamics play a critical role in all processes involved in DNA metabolism, e.g. replication, transcription, repair and recombination. Radiation is a useful tool to study the biological effects of chromatin alterations. Conversely, radiotherapy and radiodiagnosis raise questions about the influence of chromatin integrity on clinical features and secondary effects. This review focuses on the link between DNA damage and chromatin structure at different scales, showing how a comprehensive multiscale vision is required to understand better the effect of radiations on DNA. Clinical aspects related to high- and low-dose of radiation and chromosomal instability will be discussed. At the same time, we will show that the analysis of the radiation-induced DNA damage distribution provides good insight on chromatin structure. Hence, we argue that chromatin "structuralists" and radiobiological "clinicians" would each benefit from more collaboration with the other. We hope that this focused review will help in this regard.

Relevance and limitations of crowding, fractal, and polymer models to describe nuclear architecture.

Chromosome architecture plays an essential role for all nuclear functions, and its physical description has attracted considerable interest over the last few years among the biophysics community. These researches at the frontiers of physics and biology have been stimulated by the demand for quantitative analysis of molecular biology experiments, which provide comprehensive data on chromosome folding, or of live cell imaging experiments that enable researchers to visualize selected chromosome loci in living or fixed cells. In this review our goal is to survey several nonmutually exclusive models that have emerged to describe the folding of DNA in the nucleus, the dynamics of proteins in the nucleoplasm, or the movements of chromosome loci. We focus on three classes of models, namely molecular crowding, fractal, and polymer models, draw comparisons, and discuss their merits and limitations in the context of chromosome structure and dynamics, or nuclear protein navigation in the nucleoplasm. Finally, we identify future challenges in the roadmap to a unified model of the nuclear environment.

A fractal model for nuclear organization: current evidence and biological implications.

Chromatin is a multiscale structure on which transcription, replication, recombination and repair of the genome occur. To fully understand any of these processes at the molecular level under physiological conditions, a clear picture of the polymorphic and dynamic organization of chromatin in the eukaryotic nucleus is required. Recent studies indicate that a fractal model of chromatin architecture is consistent with both the reaction-diffusion properties of chromatin interacting proteins and with structural data on chromatin interminglement. In this study, we provide a critical overview of the experimental evidence that support a fractal organization of chromatin. On this basis, we discuss the functional implications of a fractal chromatin model for biological processes and propose future experiments to probe chromatin organization further that should allow to strongly support or invalidate the fractal hypothesis.

ATP-independent cooperative binding of yeast Isw1a to bare and nucleosomal DNA.

Among chromatin remodeling factors, the ISWI family displays a nucleosome-enhanced ATPase activity coupled to DNA translocation. While these enzymes are known to bind to DNA, their activity has not been fully characterized. Here we use TEM imaging and single molecule manipulation to investigate the interaction between DNA and yeast Isw1a. We show that Isw1a displays a highly cooperative ATP-independent binding to and bridging between DNA segments. Under appropriate tension, rare single nucleation events can sometimes be observed and loop DNA with a regular step. These nucleation events are often followed by binding of successive complexes bridging between nearby DNA segments in a zipper-like fashion, as confirmed by TEM observations. On nucleosomal substrates, we show that the specific ATP-dependent remodeling activity occurs in the context of cooperative Isw1a complexes bridging extranucleosomal DNA. Our results are interpreted in the context of the recently published partial structure of Isw1a and support its acting as a "protein ruler" (with possibly more than one tick).

On the topology of chromatin fibres.

The ability of cells to pack, use and duplicate DNA remains one of the most fascinating questions in biology. To understand DNA organization and dynamics, it is important to consider the physical and topological constraints acting on it. In the eukaryotic cell nucleus, DNA is organized by proteins acting as spools on which DNA can be wrapped. These proteins can subsequently interact and form a structure called the chromatin fibre. Using a simple geometric model, we propose a general method for computing topological properties (twist, writhe and linking number) of the DNA embedded in those fibres. The relevance of the method is reviewed through the analysis of magnetic tweezers single molecule experiments that revealed unexpected properties of the chromatin fibre. Possible biological implications of these results are discussed.

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.

Nucleosome-remodelling machines and other molecular motors observed at the single-molecule level.

Through its capability to transiently pack and unpack our genome, chromatin is a key player in the regulation of gene expression. Single-molecule approaches have recently complemented conventional biochemical and biophysical techniques to decipher the complex mechanisms ruling chromatin dynamics. Micromanipulations with tweezers (magnetic or optical) and imaging with molecular microscopy (electron or atomic force) have indeed provided opportunities to handle and visualize single molecules, and to measure the forces and torques produced by molecular motors, along with their effects on DNA or nucleosomal templates. By giving access to dynamic events that tend to be blurred in traditional biochemical bulk experiments, these techniques provide critical information regarding the mechanisms underlying the regulation of gene activation and deactivation by nucleosome and chromatin structural changes. This minireview describes some single-molecule approaches to the study of ATP-consuming molecular motors acting on DNA, with applications to the case of nucleosome-remodelling machines.

Linker histones incorporation maintains chromatin fiber plasticity.

Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones.

Conformational transition of DNA bound to Hfq probed by infrared spectroscopy.

Hfq is a bacterial protein involved in RNA metabolism. Besides this, Hfq's role in DNA restructuring has also been suggested. Since this mechanism remains unclear, we examined the DNA conformation upon Hfq binding by combining vibrational spectroscopy and neutron scattering. Our analysis reveals that Hfq, which preferentially interacts with deoxyadenosine rich sequences, induces partial opening of dA-dT sequences accompanied by sugar repuckering of the dA strand and hence results in a heteronomous A/B duplex. Sugar repuckering is probably correlated with a global dehydration of the complex. By taking into account Hfq's preferential binding to A-tracts, which are commonly found in promoters, potential biological implications of Hfq binding to DNA are discussed.

Chromatin fiber dynamics under tension and torsion.

Genetic and epigenetic information in eukaryotic cells is carried on chromosomes, basically consisting of large compact supercoiled chromatin fibers. Micromanipulations have recently led to great advances in the knowledge of the complex mechanisms underlying the regulation of DNA transaction events by nucleosome and chromatin structural changes. Indeed, magnetic and optical tweezers have allowed opportunities to handle single nucleosomal particles or nucleosomal arrays and measure their response to forces and torques, mimicking the molecular constraints imposed in vivo by various molecular motors acting on the DNA. These challenging technical approaches provide us with deeper understanding of the way chromatin dynamically packages our genome and participates in the regulation of cellular metabolism.

Intrinsic flexibility of B-DNA: the experimental TRX scale.

B-DNA flexibility, crucial for DNA-protein recognition, is sequence dependent. Free DNA in solution would in principle be the best reference state to uncover the relation between base sequences and their intrinsic flexibility; however, this has long been hampered by a lack of suitable experimental data. We investigated this relationship by compiling and analyzing a large dataset of NMR (31)P chemical shifts in solution. These measurements reflect the BI <--> BII equilibrium in DNA, intimately correlated to helicoidal descriptors of the curvature, winding and groove dimensions. Comparing the ten complementary DNA dinucleotide steps indicates that some steps are much more flexible than others. This malleability is primarily controlled at the dinucleotide level, modulated by the tetranucleotide environment. Our analyses provide an experimental scale called TRX that quantifies the intrinsic flexibility of the ten dinucleotide steps in terms of Twist, Roll, and X-disp (base pair displacement). Applying the TRX scale to DNA sequences optimized for nucleosome formation reveals a 10 base-pair periodic alternation of stiff and flexible regions. Thus, DNA flexibility captured by the TRX scale is relevant to nucleosome formation, suggesting that this scale may be of general interest to better understand protein-DNA recognition.