Biochimie 91 (2009) 943–950

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Meeting report

Nucleic acid–protein interactions: Wedding for love or circumstances? Christophe Lavelle a, *, Malcolm Buckle b a b

Interdisciplinary Research Institute, CNRS USR 3078, 50 Av Halley, Parc Scientifique de la Haute Borne, F-59655 Villeneuve d’Ascq, France LBPA, CNRS UMR 8113, ENS Cachan, F-94235 Cachan, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 March 2009 Accepted 16 April 2009 Available online 5 May 2009

The sixth Figeac meeting on nucleic acid–protein interactions was held in Figeac, France, from September 26th to October 1st, 2008. It was organized by the working group ‘‘nucleic acid–protein interactions and gene expression’’ from the French Society for Biochemistry and Molecular Biology. This report briefly summarizes the presentations by 40 speakers during the four plenary sessions, which were organised as follows: (1) nucleic acids: targets and tools, (2) RNA superstar, (3) nuclear structure and dynamics, and (4) new concepts – new approaches. A total of 22 plenary lectures, 18 oral communications and 40 posters were presented over the 5 days, providing a highly stimulating environment for scientific exchange between the w80 participants (biochemists, physicists, bio-informaticians and molecular and cellular biologists). Ó 2009 Elsevier Masson SAS. All rights reserved.

Keywords: Nucleic acids DNA RNA Chromatin

The Figeac sessions, kindly hosted by the city of Figeac (southwest of France), take place every 2 years and assemble the French nucleic acids and protein community in the enjoyable surroundings of the Lot valley. This 6th edition started with a keynote lecture by Marcel Me´chali (Institut de Ge´ne´tique Humaine, Montpellier), who provided an overview of the programmed assembly of replication origins during cell cycle and differentiation (Fig. 1). Notably, Me´chali presented two recent mechanistic insights into the control of replication: first, he demonstrated that MCM9 forms a stable complex with the licensing factor Cdt1 and that its binding to chromatin is required for the recruitment of the MCM2-7 helicase [1]. He then showed that topoisomerase II couples DNA replication termination with the clearing of replication complexes in order to reset replicons at mitosis [2]. Me´chali finally presented results showing that origin specification in higher eukaryotes is not strict (or at least not as clearly defined as in Escherichia coli or Saccharomyces cerevisiae), and can be reprogrammed during development [3]. More generally, DNA replication origins might rely on epigenetic mechanisms that would reflect the need to establish and control different programmes of gene expression during the development of complex organisms [4]. 1. Nucleic acids: targets and tools This session, chaired by J.F. Riou, T. Garestier-He´le`ne and M. Me´chali, began with Sonia Da Rocha Gomes (Inserm U869,

* Corresponding author. Tel.: þ33 6 24 71 44 03. E-mail address: [email protected] (C. Lavelle). 0300-9084/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.04.017

University of Bordeaux) who presented new developments in aptamer applications. Aptamers are RNA or DNA oligonucleotides isolated from a randomly synthesized library through an in vitro selection procedure (SELEX – Systematic Evolution of Ligands by EXponential enrichment). They are thus attractive as exquisite tools for functional genomics analysis and as promising prototypes for therapeutic agents [5]. Da Rocha Gomes and co-workers have recently identified, selected and chemically optimised a new aptamer that can target a biomarker of tumour growth, the MMP-9 extracellular matrix protein. Kenza Lahkim Bennani-Belhaj (Institut Curie, CNRS UMR 2027, Orsay) determined, at the cellular level, the respective contributions of BLM and RAD51 in preventing genomic instability, proposing a model in which these two proteins are required to restart the replication of particular DNA structures, such as fragile sites for example. Vale´rie Borde (Institut Curie, CNRS UMR 7147, Paris) addressed the distribution of recombination events along meiotic chromosomes in budding yeasts. Meiotic recombination is initiated by the formation of programmed DNA double-strand breaks (DSBs) catalyzed by the SpoII protein. By examining the genome-wide binding and cleavage of the Gal4 DNA binding domain (Gal4BD)-SpoII fusion protein, Borde and colleagues showed that the targeting of SpoII to chromatin leads to both local stimulation and genomewide redistribution of recombination initiation [6]. Furthermore, SpoII cleavage sites are found close to H3K4 tri-methylated histones. However, although this modification is usually associated with an ‘‘active’’ chromatin state [7], no correlation was found between DSBs location and gene expression. In a set1D mutant, in the absence of H3K4 tri-methylation, DSB formation is strongly

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Fig. 1. DNA replication visualized by DNA combing of single molecules. Replicating DNA is marked by two consecutive pulses, one with IdU followed by one with CldU, and the fluorescence detected using corresponding antibodies. DNA is then extracted and combed on silanized glasses. Different combed DNA molecules are shown, illustrating the initiation of DNA replication and the progression of the replication fork in a bidirectional manner (Stanojcik and Me´chali, unpublished data).

reduced, suggesting that this histone mark might be locally recruiting meiotic DSB forming factors [8]. Marie-Jose`phe Giraud-Panis (Laboratoire Joliot Curie, CNRS UMR 5161, Lyon) presented novel mechanistic insights into chromosomal end protection. Invasion of the 30 overhang into duplex DNA has been proposed to fold telomeres into t-loops, this process being facilitated in vitro by the telomeric protein TRF2. Using a variety of TRF2 mutants, Giraud-Panis and colleagues revealed that this protein generates positive supercoiling in DNA and that this topological activity correlates with its ability to stimulate strand invasion. This suggests that TRF2 complexes, by constraining DNA around themselves in a right-handed conformation, could induce untwisting within telomeric sequences and favour strand invasion [9]. Carine Giovannangeli (Museum National d’Histoire Naturelle, CNRS UMR 5153, Paris) reported that conjugates of a DNA cleaving molecule (orthophenanthroline; OP) and triplex-forming oligonucleotides (TFOs) are synthetic nucleases efficient at stimulating targeted genome modification. TFOs, which bind specific DNA sequences via Hoogsteen hydrogen bonds, have already been used to induce targeted mutagenesis in model systems [10]. To overcome the relative inefficiency of gene modification by homologous recombination, a DSB can be introduced into the target. Giovannangeli and colleagues showed that in cultured cells, OP–TFO conjugates induce targeted DSBs and that mutations at the target site were found in approximately 10% of treated cells [11]. Such synthetic sequence-specific nucleases therefore constitute a promising alternative to protein-based endonucleases for targeted gene modification. Olivier Hyrien (Ecole Normale Supe´rieure, CNRS UMR 8541, Paris) investigated DNA replication timing in Xenopus egg extracts. DNA combing, kinetic modelling and other studies on this system have suggested that potential origins are much more abundant than actual initiation events and that the time-dependent rate of initiation increases through the S phase to ensure the rapid completion of unreplicated gaps [12]. To quantitatively and robustly account for the observed changes in initiation rate and fork density, Hyrien and colleagues recently proposed a model that combines time-dependent changes in availability of a replication factor and a fork-density dependent affinity of this factor for potential origins [13]. Interestingly, Hyrien also showed experimental results suggesting that, in this embryonic system, replication timing is deterministic at the scale of large chromatin domains

(1–5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50–100 kb) [14]. Marcello Nollmann (Centre de Biochimie Structurale, CNRS UMR 5048, Montpellier) studied the properties of DNA translocation by SpoIIIE, an AAA þ ATPase responsible for intercompartmental chromosome translocation in sporulating bacteria and highly homologue to FtsK (Escherichia coli). While SpoIIIE has been suggested to be a unidirectional DNA transporter that exports DNA from the compartment in which it assembles, FtsK was shown to establish translocation directionality by interacting with highly skewed chromosomal sequences. Based on single molecule, bioinformatics and in vivo fluorescence microscopy data, Nollmann and colleagues proposed a novel sequence-directed DNA exporter mechanism that reconciles all previous data and constitutes a unified model for directional DNA transport by this family of AAA þ ring ATPases [15]. Audrey Olivier (Laboratoire de Microbiologie et Ge´ne´tique Mole´culaires, CNRS UMR 5100, Toulouse) concluded this session by showing preliminary results on the respective roles of SsbA and SsbB, two single strand binding (SSB) proteins present in Streptococcus pneumoniae. During genetic transformation of the Grampositive bacterium S. pneumoniae, single strands from native donor DNA enter competent cells, where they associate with SsbB [16]. The SsbA protein is similar in size to the well-characterized SSB protein from E. coli, whereas the SsbB protein is a smaller protein that is specifically induced during natural transformation and has no counterpart in E. coli. The exact role of this last protein remains to be identified. 2. Superstar RNA This session, chaired by M. Amor-Gueret, F. Lejeune, S. Kochbin and L. Ponchon, started with a talk by Fabrice Lejeune (Institut Pasteur, Lille) who recently identified an inhibitor of the nonsensemediated mRNA decay (NMD) pathway in mammals. NMD is a quality-control mechanism that degrades mRNA harbouring a premature termination codon to prevent the synthesis of truncated proteins. Lejeune and colleagues demonstrated that a small molecule, called NMD inhibitor 1 (NMDI 1), induced the loss of interactions between two partners of the NMD pathway, hSMG5 and hUPF1, leading to stabilization of hyper-phosphorylated isoforms of hUPF1. Incubation of cells with NMDI 1 showed that NMD factors and mRNAs subject to NMD, transit through processing bodies (P-bodies), suggesting a model in which mRNA and NMD factors are sequentially recruited to P-bodies [17]. Ve´ronique Arluison (Institut de Biologie Physico-Chimique, CNRS UPR 9073, Paris) showed that a natural bacterial RNA, DsrA, has the property to auto-assemble. This small non-coding RNA (ncRNA) of E. coli controls the translation and turnover of mRNA including rpos mRNA involved in bacterial stress response. This regulation is mediated by Hfq [18], an RNA chaperone able to polymerize into well-ordered fibrillar structures [19]. Strikingly, Arluison and colleagues showed that DsrA by itself could also form long polymers, a property apparently provided by a minimal region of 22 nucleotides (Fig. 2) [20a]. This is the first time that such structures have been observed with a natural RNA. This polymerization is suggested to be involved in the regulation of DsrA ncRNA concentration in vivo. More strikingly, evidence was presented that DsrA is not an isolated case of natural polymerizing RNA, which means ncRNA auto-assembly may be a general mechanism involved in different cellular processes [20b]. Herve´ Le Hir (Centre de Ge´ne´tique Mole´culaire, CNRS UPR 2167, Gif-sur-Yvette) presented recent advances in the structural characterization of the exon junction complex (EJC) [21]. In higher eukaryotes, EJC is loaded onto spliced mRNAs at a precise position

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Fig. 2. Small non-coding RNA visualized by atomic force microscopy (AFM). A 22 nt chemically synthesized sequence from DsrA (an 87 nt E. coli ncRNA) auto-assembles, forming long (up to 500 nm) polymers; note that some of the polymers have their ends coming into contact, forming circles (bar 250 nm; Pie´trement and Arluison, unpublished data).

upstream of exon junctions, where it remains during nuclear export and cytoplasmic localisation until removal during the first translation round. The EJC core complex consists of four proteins (eIF4AIII, MAGOH, Y14 and MLN51) that form a dynamic binding platform for a variety of peripheral factors involved in mRNA metabolism. A crystal structure of this tetrameric complex was obtained in the presence of ATP and RNA [22], providing Le Hir and colleagues with an important starting point to elucidate the molecular mechanisms of the multiple EJC functions. Xavier Manival (MAEM, UMR 7567 CNRS-UHP-Nancy I) showed structural insights into the biogenesis of eukaryotic ribonucleoprotein particles (RNPs) containing L7Ae-like proteins. These RNAbinding proteins (such as 15.5 kDa/Snu13p) play an essential role in the assembly, structure and function of several RNPs, including box C/D and H/ACA snoRNPs, U4 snRNP and telomerase. Recent studies in Manival’s group and others showed that, surprisingly, the assembly of these RNPs requires several factors in addition to mature particle proteins such as the protein NUFIP/Rsa1p [23]. By using site-directed mutagenesis, Manival and colleagues identified determinants for the formation of a tripartite complex containing: an RNA molecule with the U3 B/C motif, Snu13p and the yPEP fragment of Rsa1. They consequently used computer modelling to construct a 3D model of this complex, consistent with the experimental data (Fig. 3). Fabien Darfeuille (INSERM U869, Universite´ de Bordeaux) presented some ongoing work about the regulatory role of small RNAs in Helicobacter pylori. This human pathogen colonizes the harsh environmental niche of the stomach, causing, among other things, gastric ulcers and cancer. The mechanisms of regulation of gene expression in this bacterium are largely unknown. Genomic research has recently led to an explosion in the identification of new sRNAs in E. coli and other pathogenic bacteria. It has been proposed that many of these sRNA could be regulators of target mRNAs under certain environmental growth conditions, in particular environmental stress conditions. Darfeuille and colleagues are

Fig. 3. A 3D molecular surface representation of Snu13p–yPEP interaction model established by homology modeling and molecular docking. The interaction between the protein Snu13p and the peptide yPEP is made by the molecular clamp formed by yPEP residues E18 and R20. The 3D structure of yPEP peptide was build by homology modeling using the atomic coordinates of the HMG domain of the FACT factor (PDB ID : 1WXL). The rigid-body docking refinement procedure used with the X-ray structure of Snu13p protein (PDB ID : 2ALE) minimizes electrostatic and van der Waals energies of interaction between the two partners (Manival, unpublished data).

now working on the identification of sRNA in H. pylori that are specifically induced or repressed during the colonization of the stomach (acidic stress) or in contact with the host epithelial gastric cells. Sylvie Auxilien (Laboratoire d’Enzymologie et Biochimie Structurales, CNRS UPR 3082, Gif-sur-Yvette) presented work on the specificity of two S-adenosyl-L-methionine dependent RNA(uracil,C5)-methyltransferases from Pyrococcus abyssi. Amongst the methyltransferase family, the sequences of these two are most closely related to E. coli RumA, a protein catalyzing the formation of 5-methyluridine (m5U) at position 1939 in E. coli 23S rRNA. Unexpectedly, none of the two P. abyssi enzymes displays RumA-like specificity but rather they catalyze m5U formation either in tRNA [24] or at position 747 in 23S rRNA. These results, together with phylogenetic analysis, suggest that the corresponding genes have been acquired via a single horizontal gene transfer of a RumA-type gene from a bacterial donor into the common ancestor of some Archaea. This transfer event was followed by gene duplication leading to two closely related genes and at least one change of target specificity. Lluis Ribas de Pouplana (Barcelona Institute for Research in Biomedicine, Barcelona) presented new insights into an established but still fascinating problem: tRNA recognition and specificity In this context, the assignment of AUG codons to methionine remains

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a central question concerning the evolution of the genetic code. De Pouplana and colleagues have shown that Mycoplasma penetrans methionyl-tRNA synthetase can directly differentiate between tRNA(Ile) (CAU) and tRNA(Met) (CAU) transcripts (a recognition normally achieved through the modification of anticodon bases), this discrimination mechanism being based only on interactions with the acceptor stems of the two tRNA. This remarkable observation suggests that in certain species, the fidelity of translation of methionine codons requires a discrimination mechanism that is independent of the information contained in the anticodon [25]. Magali Blaud (Laboratoire de Maturation des ARN et Enzymologie Mole´culaire, CNRS UMR 7567, Vandoeuvre-les-Nancy) discussed the role of the aNOP10–L7Ae interaction in small ribonuclear particle (sRNPs) assembly and activity. Archael box H/CA sRNPs are involved in the conversion of uridine to pseudouridine in RNA. Each particle contains one guide sRNA and four core proteins: aCBF5, aNOP10, L7Ae and aGAR1. aNOP10 is required for association of the target RNA with assembled sub-RNPs [26] and for the correct positioning of residues in the aCBF5 catalytic centre [27]. The results of Blaud and colleagues suggest that one important function of L7Ae, through its interaction with aNOP10, is to promote the formation of an RNP architecture that is optimal for the pseudouridylation activity of archaeal box H/CA sRNPs. Klaus Scherrer (Institut Jacques Monod, Paris) ended this session with an attempt to clarify the definition of a gene in order to distinguish what the gene is coding for (namely a specific polypeptide), how gene expression occurs and how it is controlled. It is becoming increasingly clear that the relationship between the information stored at the level of DNA and its functional products are very intricate, and that regulatory aspects are as important and essential as the basal information coding for the products. To clearly separate product and regulative information, while keeping the fundamental relation between coding and function, Klaus has coined the new term ‘‘genon’’, which relates to the program regulating the expression of a gene, superimposed in cis onto the coding sequence, the program being implemented by RNA–protein (mRNPs) and RNA–RNA (RNAi) interactions. This concept is advanced as an attempt to provide a clearer comprehension of the nature of genes and gene expression supported by a new information theory model [28,29]. 3. Nuclear structure and dynamics This session, chaired by C. Lavelle, A. Taddei and O. Hyrien opened with a presentation by Angela Taddei (Institut Curie, CNRS UMR 218, Paris) who stressed the active role played by nuclear organization in gene regulation. A key component of the nuclear architecture is the nuclear envelope, studded with nuclear pore complexes that serve as gateways for communication between the nucleoplasm and cytoplasm. In budding yeast, telomeres are anchored at the nuclear envelope, forming foci that sequester silent information regulators (SIR factors). Taddei and colleagues have shown that the efficiency of subtelomeric gene repression depends not only on the strength of SIR factor recruitment, but also on the accumulation of SIRs in perinuclear foci, which result from the clustering of telomeres [30]. Remarkably, although the nuclear periphery has traditionally been described as a repressive compartment and repository for gene-poor chromosome regions, recent studies in yeast have demonstrated that both repressive and activating domains can be found at the periphery of the nucleus and that association with the nuclear envelope can even favour the expression of particular genes [31]. Other studies carried out on mammalian cells confirmed that the nuclear periphery is not refractory to gene transcription but can modulate the activity of certain genes [32]. The 3D organization of the genome thus seems

to provide an additional level of regulation shared by all eukaryotes to finely tune gene expression. Guy Cathala (Institut de Ge´ne´tique Mole´culaire, CNRS UMR 5535, Montpellier) discussed chromosome conformation capture (3C) technology, a pioneering methodology that allows in vivo exploration of genomic organization at a scale encompassing tens to hundreds of kilo base-pairs. 3C technology involves formaldehyde treatment of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are cross-linked in the various cell populations. Cathala and colleagues recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method (named 3C-qPCR) that more accurately determines cross-linking frequencies than current semi-quantitative 3C strategies (that essentially rely on measuring the intensity of ethidium bromidestained PCR products separated by gel-electrophoresis) [33]. This technique has recently been applied to the study of different loci, including Igf2/H19 and Dlk1/Gtl2 [34]. Sylvie Rimsky (Ecole Normale Supe´rieure de Cachan, CNRS UMR 8113, Cachan) presented recent data on the role of H–NS, a protein of the bacterial nucleoid involved in DNA compaction and transcription regulation. In vivo, H–NS selectively silences specific genes of the bacterial chromosome but the molecular basis for its selectivity remained poorly understood [35]. Rimsky and colleagues have shown that the negative regulatory element of the supercoiling-sensitive E. coli proU gene contains two identical highaffinity binding sites for H–NS and that cooperative binding of H–NS is abrogated by changes in DNA superhelical density, temperature and removal of these sites. Synergy between these sites and lower affinity sites in the region favours the formation of a specific nucleoprotein complex that efficiently silences transcription at proU (and presumably at other genes) [36]. Comparison of this 10 bp high-affinity binding motif with other predicted and confirmed H–NS sites allowed the identification of a consensus recognition sequence for H–NS. The discovery of a specific binding site for H–NS and the involvement of cooperativity in the selection of topologically sensitive DNA structures grant new insights into the potential mechanism of H–NS–DNA interactions and bacterial chromatin protein interactions in general in the regulation of prokaryotic gene expression. Claire Rosnoblet (Institut de Recherche Interdisciplinaire, CNRS USR 3078, Villeneuve d’Ascq) presented some ongoing work on the interaction of histone methyltransferase SUV420H2 and the hetorochromatin-associated protein HP1. Histone lysine methylation is involved in many biological processes such as heterochromatin formation, chromosome X inactivation, genomic imprinting and transcriptional regulation [37]. SUV420H2 is responsible for H4K20 tri-methylation, an epigenetic marker usually associated with pericentric heterochromatin. Rosnoblet and colleagues have recently identified HP1 as a major partner of SUV420H2 through interactions with its chromo-shadow domain. Furthermore, FRAP experiments showed that HP1 is much more mobile in the nucleus than SUV420H2, suggesting that these two proteins interact dynamically while SUV420H2 is bound to the chromatin. Christophe Lavelle (Institut Curie, CNRS UMR 168, Paris) addressed different issues in eukaryotic chromatin structure and dynamics. First, he described many aspects of nucleosome polymorphism (histone variants, histone tails modifications, sequence properties of wrapped DNA, nucleosomal subparticles), thus illustrating that the idea of a so-called ‘‘canonical’’ nucleosome is clearly an oversimplification. [38]. Second, he discussed how nucleosomal properties themselves influenced chromatin fibre structure and its response to physical constraints [39]. Third, Lavelle emphasized the critical role of DNA in establishing particular chromatin folding, confirming that the DNA sequence encodes important information

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concerning local chromatin properties [40]. Finally, he described various mechanisms of chromatin remodeling, taking as an example the RSC remodeler (Fig. 4). Lavelle and colleagues also showed that Rad51, the well-known protagonist of homologous recombination in eukaryotic cells, had a powerful chromatin remodeling effect. Indeed, Rad51 polymerization displaced nucleosomes in front of the progressing filament along considerable stretches of the DNA [41]. This opens up new possibilities for understanding DNA recombination and reveals at the same time a new type of ATP-dependent chromatin remodeling mechanism. Yegor Vassetzky (Institut Gustave Roussy, CNRS UMR 8126, Villejuif) presented some recent results obtained on mantle cell lymphoma (MCL), a non-Hodgkin lymphoma with one of the worst prognoses amongst all known lymphomas. This lymphoma is characterized by the deregulated expression of cyclin D which is targeted by the t(11;14) (q13;q32) chromosomal translocation, the genetic hallmark of the disease [42]. Vassetzky and colleagues made the hypothesis that this translocation may induce a re-localization of the 11q13 fragment from the periphery (where it stands in a mostly heterochromatic environment) to a more central (euchromatic) region of the nucleus. Indeed, they were able to detect a displacement of the 11q13 region towards the centre of the nucleus (more specifically in the vicinity of the nucleolus) in MCL lymphocytes. This led them to propose that the translocation of the 11q13 region close to the nucleolus (a very active region where rRNA genes, in particular found on chromosome 14, are transcribed) could explain the transcriptional de-regulation observed in MCL (Allinne et al. to be submitted). Audrey Costes (Laboratoire de Microbiologie et Ge´ne´tique Mole´culaires, CNRS UMR 5100, Toulouse) discussed the response to DNA replication arrest in bacteria, which generally culminates in the reassembly of the replisome on inactivated forks in order to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a remodeling of the arrested fork. Costes and colleagues recently reported that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo [43]. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a ‘‘repair toolbox’’ with active replication forks. Several other partners

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belonging to the ‘‘interactome’’ of SSB have been identified and their function is currently under investigation. Saadi Khochbin (Institut Albert Bonniot, INSERM U823, Grenoble) presented some recent insights into the stepwise replacement of histones by transition proteins and protamines during male germ cell maturation. The mechanisms directing this post-meiotic massive genome reorganisation are quite poorly characterized [44]. Khochbin and colleagues recently found new testis-specific histone variants that accumulate essentially during late spermatogenesis and are maintained in association with the genome of mature spermatozoa [45]. Furthermore, they showed that this late event follows a general genome reorganisation triggered by a massive wave of histone acetylation occurring at the beginning of the spermatid elongation process. Iain Campuzano (Waters Corporation, Manchester) concluded this session by presenting a rapid method for determining the shape and cross sections of macromolecular complexes. By coupling mass spectrometry with ion mobility separation (IMS), both the intact quaternary mass and the collision cross section of biological macromolecules can be accurately measured. This approach has been applied to tryptophan RNA-binding protein (TRAP), providing definitive evidence that protein quaternary structure can be maintained in the absence of bulk water and highlighting the potential of ion mobility separation for defining shapes of heterogeneous macromolecular assemblies [46]. 4. New concepts – new approaches This session, chaired by C. Royer, L. Letellier, M. Buckle and M. Kochoyan, was a joint session with the French Biophysical Society annual meeting. It started with Carmelo Di Primo (Institut Europe´en de Chimie et Biologie, INSERM U869, Pessac) who demonstrated the utility of surface plasmon resonance (SPR) to systematically analyse RNA–protein and RNA–RNA interactions. Ternary complexes formed for example between a loop–loop RNA complex and a specific stabilising protein, as well as RNA–RNA interactions can be characterized [47]. SPR was used to demonstrate that inter backbone hydrogen bonds observed in the X-ray crystallographic model of the HIV-1 TAR-RNA aptamer complex structure were crucial non-canonical interactions for complex stability [48]. This technique also appears particularly useful for efficient aptamer screening, as exemplified by Di Primo and

Fig. 4. Nucleosome remodeling by RSC. RSC (Remodels the Structure of Chromatin) is an essential, abundant ATP-dependent remodeling factor acting in various cellular processes such as DNA transcription and repair. Although the precise mechanism by which RSC destabilizes histone–DNA interactions and alters nucleosome positioning is not known, a lot of data suggests that it does so by creating a loop at the edge of the nucleosome, which then propagates in a wave-like manner at the surface of the histone octamer (nucleosome before (a), during (b) and after (c) interaction with RSC; bar 40 nm; Lavelle and Le Cam, unpublished data).

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colleagues who characterized modified aptamers targeted to the TAR-RNA element of HIV-1 [49]. Emmanuel Margeat (Centre de Biochimie Structurale, CNRS UMR 5048, Montpellier) showed how single molecule fluorescence studies shed light on molecular mechanisms of transcription [50,51]. Specifically Margeat and colleagues re examined the process of escape from initiating transcription complexes. Jose´phine Abi Ghanem (Laboratoire de Biochimie The´orique, CNRS UPR 9080, Paris) discussed the influence of monovalent cations on DNA conformation. Indeed, a recent NMR study of DNA with either Naþ or Kþ at physiological concentrations revealed that the interaction of these ions with DNA are different and sequence dependent [52]. Hence, ions finely modulate the intrinsic properties of DNA, and thus intriguingly possibly impact on DNA packaging and readout. Françoise Paquet (Centre de Biophysique Mole´culaire, CNRS UPR 4301, Orle´ans) presented structural and dynamic NMR studies on MC1, a chromosomal protein involved in the DNA compaction of some methanogenic Archaea [53] (Fig. 5). This small monomeric protein sharply bends DNA and is able to discriminate between different deformations of the DNA helix. New structural details coupled with recent dynamic observations of the MC1–DNA interaction were obtained that match former results obtained using chemical probes [54]. Luc Ponchon (Laboratoire de Cristallographie et de RMN Biologique, CNRS UMR 8015, Paris) described a generic approach for expressing and purifying structured RNA in E. coli, using tools that parallel those available for recombinant proteins. This system is based on a camouflage strategy, the ‘‘tRNA scaffold’’, in which recombinant RNA is disguised as a natural RNA and thus hijacks the host machinery, escaping cellular RNases [55]. Eric Ennifar (Institut de Biologie Mole´culaire et Cellulaire, CNRS UPR 9002, Strasbourg) discussed aminoglycoside binding to the HIV-1 dimerization initiation site. Aminoglycosides increase the stability and the melting temperature of the loop–loop interaction and block the conversion from kissing-loop complex to extended duplex [56]. These features proved useful for selecting new molecules with improved specificity and affinity towards the HIV-1 DIS RNA [57]. Following Ennifar’s talk, a plenary conference was given by Fre´de´ric Dardel (Laboratoire de Cristallographie et de RMN Biologique, CNRS UMR 8015, Paris) for the account of the French Biophysical Society meeting. Dardel presented recent studies on the thermophile Aquifex aeolicus which uses molecular hydrogen as an energy source and oxygen as a final electron acceptor. Dardel and colleagues were able to purify and then synthesize a supercomplex consisting of a membrane associated sulphur hydrogen

Fig. 5. 3D structure of the archaeal protein MC1. This small monomeric protein has been shown to sharply bend DNA [53].

reductase that couples periplasmic hydrogen oxidation with cytoplasmic reduction of elemental sulphur. This super-complex consisted of a membrane hydrogenase II and a sulphur reductase made up of 3 separate subunits, several Fe–S clusters and a molybdene co-factor. The group demonstrated the novel involvement of a quinine group in electron transfer within the super-complex and then went on to solve the 3D structure using NMR. This novel and original observation of the role of quinines in such complexes has lead to an ongoing pursuit not only of potential partners of this super-complex but also on what is the role of lipids in complex stability in A. aeolicus. Bianca Sclavi (Ecole Normale Supe´rieure de Cachan, CNRS UMR 8113, Cachan) discussed how to perform efficient quantitative characterization of the large number of macromolecular interactions present in cellular regulatory networks. Bianca stressed the importance of rapid interrogation techniques (e.g. synchrotron generated hydroxyl radical DNA cleavage) having high spatial and temporal resolution. Bianca also described novel applications of capillary electrophoretic gel separation techniques and analysis that allowed accurate kinetic quantification. By re-examining existing models for the formation of transcription complexes Bianca was able to demonstrate that such models were indeed oversimplified and that dynamic approaches opened exciting avenues for new paradigm models. Bruno Klaholz (IGBMC, Illkirch) described the interplay of mRNA and factors in intermediate states of the initiating protein synthesis machinery. By using a combination of biochemistry, cryoelectron microscopy, crystallography and bioinformatics, Klaholz and colleagues addressed the mechanism by which mRNA and initiation factors regulate the ribosome activity [58,59]. These studies reveal the role of mRNA structure in translation initiation regulation, in particular the mechanism of transient ribosomeentrapment by an mRNA whose folded state is stabilized by a repressor protein. Sequence and structure analysis suggested the existence of a conserved site on the ribosome for binding regulatory mRNAs [58]. The structure of the 30S translation initiation complex was also presented [59], revealing how initiation factors stabilise the initiator tRNA in the first gene-decoding complex within the protein synthesis process, thereby visualizing an intermediate complex before the ribosome machinery fully assembles and starts protein synthesis (Fig. 6). Philippe Dumas (IBMC, CNRS UPR 9002, Strasbourg) analyzed the folding kinetics of the aptamer domain of the Arabidopsis thaliana riboswitch upon binding of the pyrophosphate derivative of thiamine (ThiPP). This riboswitch, located in the 50 -UTR of an mRNA encoding a protein involved in the biosynthesis of ThiPP, ensures a negative feedback by sensing ThiPP, which leads to a structural change masking the Shine–Dalgarno sequence required for ribosome binding [60]. This is a model system since its crystallographic structure is known [61]. The Strasbourg group used hydroxyl radical footprinting (as described by B. Sclavi in the same session) for monitoring the extent of folding. They have developed gel-electrophoresis quantification software more automated in comparison to existing methods [62]. Their results show that (i) the aptamer domain is significantly pre-folded in absence of ThiPP and (ii) there exists a not yet fully understood influence of ligand concentration on the kinetics of complete folding. Jean-Marc Victor (Laboratoire de Physique The´orique de la Matie`re Condense´e, CNRS UMR 7600, Paris) showed how mechanical constraints in the nucleus could induce nucleosomal structural transitions [63,64]. Claude Nogues (Ecole Normale Supe´rieure de Cachan, CNRS UMR 8113, Cachan) introduced a novel form of surface plasmon resonance involving total imaging of the biosensor surface and

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Acknowledgements We would like to thank all the participants for their enthusiastic contribution to this meeting and also for collective help during the preparation of this report. We especially thank Mounira AmorGueret, Alain Laigle, Christian Marion, Jean-François Riou, Cathy Royer and Jean-Jacques Toulme´ for the perfect organisation of this meeting. The organizers are indebted to SFB, SFBBM, CNRS, CEA, INSERM and Re´gion Midi-Pyre´ne´es for support and Servier, Eurogentec, Wyatt and Waters for additional funding. We also greatly appreciated the efficient and kind welcome by the city of Figeac and its tourist bureau and in particular Nadine Darson for her efficiency, warmth, constant presence and help during the whole meeting. M.B. also acknowledges that the charm of the region was greatly enhanced by the presence for about 100 years of the English at a crucial moment in its history. Finally, please note that the next Figeac meeting will be held in the autumn of 2010, of course in Figeac. References [1] [2]

[3] [4] [5] [6] Fig. 6. Assembly of the 30S translation initiation complex. Linked to the small subunit of bacterial ribosome (orange), the initiation factor IF2 (green) holds the tRNA (red) bearing the first amino acid of the protein to be synthesized. Once everything is in order, the ribosome then fulfils its complete assembly (around the 3D structure: cryoTEM images of the complex) [59].

[7] [8]

[9]

addressed the optimization issue of optical/biological interface in SPR imaging. Of particular importance was the presentation of physicochemical protocols that dramatically reduced or even eradicated non-specific binding of biological molecules to biosensor surfaces allowing quantification and characterization of biomolecular interactions hitherto refractive to this approach. An exciting example of this was a quantification of the interaction of the HIV integrase with specific immobilised DNA targets. Quentin Raffy (Commissariat a` l’Energie Atomique, DSM/IRAMIS, Saclay) ended this session, and the SFBBM (‘‘Figeac’’ working group)–SFB joint meeting, by presenting a footprinting method aimed at identifying protein–protein interactions at the residue level through water radiolysis. The hydroxyl radicals produced by the water radiolysis oxidise surface residues of a protein–protein complex. The oxidised residues may be repaired in the presence of tritium, leading to radioactive labelling. The idea behind the approach is that residues involved in an intimate interaction would be less solvent-accessible and consequently shielded from hydroxyl radicals, and thus tritium incorporation would be lower. The interaction area could thus be identified by comparison of the tritium incorporation of the surface residues of the complex with that of the isolated proteins. This method has been employed with success to determine the residues of the core interaction area of a fragment of Histone 3 with human anti-silencing factor 1A (hAsf1A). More than 40 posters were also presented covering and expanding on the various issues discussed in the talks. However, due to space limitations, posters are not discussed here.

[10]

[11]

[12]

[13] [14]

[15]

[16]

[17]

[18]

[19]

M. Lutzmann, M. Mechali, MCM9 binds Cdt1 and is required for the assembly of prereplication complexes, Mol. Cell. 31 (2008) 190–200. O. Cuvier, S. Stanojcic, J.M. Lemaitre, M. Mechali, A topoisomerase IIdependent mechanism for resetting replicons at the S–M-phase transition, Genes Dev. 22 (2008) 860–865. J.M. Lemaitre, E. Danis, P. Pasero, Y. Vassetzky, M. Mechali, Mitotic remodeling of the replicon and chromosome structure, Cell 123 (2005) 787–801. M. Mechali, DNA replication origins: from sequence specificity to epigenetics, Nat. Rev. Genet. 2 (2001) 640–645. J.J. Toulme, C. Di Primo, D. Boucard, Regulating eukaryotic gene expression with aptamers, FEBS Lett. 567 (2004) 55–62. N. Robine, N. Uematsu, F. Amiot, X. Gidrol, E. Barillot, A. Nicolas, V. Borde, Genome-wide redistribution of meiotic double-strand breaks in Saccharomyces cerevisiae, Mol. Cell. Biol. 27 (2007) 1868–1880. C.B. Millar, M. Grunstein, Genome-wide patterns of histone modifications in yeast, Nat. Rev. Mol. Cell. Biol. 7 (2006) 657–666. V. Borde, N. Robine, W. Lin, S. Bonfils, V. Geli, A. Nicolas, Histone H3 lysine 4 trimethylation marks meiotic recombination initiation sites, EMBO J. 28 (2009) 99–111. S. Amiard, M. Doudeau, S. Pinte, A. Poulet, C. Lenain, C. Faivre-Moskalenko, D. Angelov, N. Hug, A. Vindigni, P. Bouvet, J. Paoletti, E. Gilson, M.J. GiraudPanis, A topological mechanism for TRF2-enhanced strand invasion, Nat. Struct. Mol. Biol. 14 (2007) 147–154. P. Simon, F. Cannata, J.P. Concordet, C. Giovannangeli, Targeting DNA with triplex-forming oligonucleotides to modify gene sequence, Biochimie 90 (2008) 1109–1116. F. Cannata, E. Brunet, L. Perrouault, V. Roig, S. Ait-Si-Ali, U. Asseline, J.P. Concordet, C. Giovannangeli, Triplex-forming oligonucleotide–orthophenanthroline conjugates for efficient targeted genome modification, Proc. Natl. Acad. Sci. U. S. A. 105 (2008) 9576–9581. O. Hyrien, K. Marheineke, A. Goldar, Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem, Bioessays 25 (2003) 116–125. A. Goldar, H. Labit, K. Marheineke, O. Hyrien, A dynamic stochastic model for DNA replication initiation in early embryos, PLoS ONE 3 (2008) e2919. H. Labit, I. Perewoska, T. Germe, O. Hyrien, K. Marheineke, DNA replication timing is deterministic at the level of chromosomal domains but stochastic at the level of replicons in Xenopus egg extracts, Nucleic Acids Res. 36 (2008) 5623–5634. J.L. Ptacin, M. Nollmann, E.C. Becker, N.R. Cozzarelli, K. Pogliano, C. Bustamante, Sequence-directed DNA export guides chromosome translocation during sporulation in Bacillus subtilis, Nat. Struct. Mol. Biol. 15 (2008) 485–493. D.A. Morrison, I. Mortier-Barriere, L. Attaiech, J.P. Claverys, Identification of the major protein component of the pneumococcal eclipse complex, J. Bacteriol. 189 (2007) 6497–6500. S. Durand, N. Cougot, F. Mahuteau-Betzer, C.H. Nguyen, D.S. Grierson, E. Bertrand, J. Tazi, F. Lejeune, Inhibition of nonsense-mediated mRNA decay (NMD) by a new chemical molecule reveals the dynamic of NMD factors in P-bodies, J. Cell. Biol. 178 (2007) 1145–1160. V. Arluison, S. Hohng, R. Roy, O. Pellegrini, P. Regnier, T. Ha, Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA, Nucleic Acids Res. 35 (2007) 999–1006. V. Arluison, C. Mura, M.R. Guzman, J. Liquier, O. Pellegrini, M. Gingery, P. Regnier, S. Marco, Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins, J. Mol. Biol. 356 (2006) 86–96.

950 [20]

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

[29] [30]

[31] [32]

[33]

[34]

[35] [36]

[37] [38] [39] [40] [41]

[42]

C. Lavelle, M. Buckle / Biochimie 91 (2009) 943–950 (a). B. Cayrol, F. Geinguenaud, J. Lacoste, F. Busi, J. Le Derout, O. Pietrement, E. Le Cam, P. Reigner, C. Lavelle, V. Arluison. Auto-assembly of E. Coli DsrA small noncoding RNA: molecular characteristics and functional consequences. RNA Biol. 6, in press; (b). F. Busi, B. Cayrol, C. Lavelle, J. Le Derout, O. Pietrement, E. Le Cam, F. Geinguenaud, J. Lacoste, P. Regnier, V. Arluison, Auto-assembly as a new regulatory mechanism of noncoding RNA, Cell Cycle 8 (2009) 952–954. H. Le Hir, G.R. Andersen, Structural insights into the exon junction complex, Curr. Opin. Struct. Biol. 18 (2008) 112–119. C.B. Andersen, L. Ballut, J.S. Johansen, H. Chamieh, K.H. Nielsen, C.L. Oliveira, J.S. Pedersen, B. Seraphin, H. Le Hir, G.R. Andersen, Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA, Science 313 (2006) 1968–1972. S. Boulon, N. Marmier-Gourrier, B. Pradet-Balade, L. Wurth, C. Verheggen, B.E. Jady, B. Rothe, C. Pescia, M.C. Robert, T. Kiss, B. Bardoni, A. Krol, C. Branlant, C. Allmang, E. Bertrand, B. Charpentier, The Hsp90 chaperone controls the biogenesis of L7Ae RNPs through conserved machinery, J. Cell. Biol. 180 (2008) 579–595. J. Urbonavicius, S. Auxilien, H. Walbott, K. Trachana, B. Golinelli-Pimpaneau, C. Brochier-Armanet, H. Grosjean, Acquisition of a bacterial RumA-type tRNA(uracil-54, C5)-methyltransferase by Archaea through an ancient horizontal gene transfer, Mol. Microbiol. 67 (2008) 323–335. T.E. Jones, C.L. Brown, R. Geslain, R.W. Alexander, L. Ribas de Pouplana, An operational RNA code for faithful assignment of AUG triplets to methionine, Mol. Cell. 29 (2008) 401–407. B. Charpentier, S. Muller, C. Branlant, Reconstitution of archaeal H/ACA small ribonucleoprotein complexes active in pseudouridylation, Nucleic Acids Res. 33 (2005) 3133–3144. S. Muller, J.B. Fourmann, C. Loegler, B. Charpentier, C. Branlant, Identification of determinants in the protein partners aCBF5 and aNOP10 necessary for the tRNA: Psi55-synthase and RNA-guided RNA: Psi-synthase activities, Nucleic Acids Res. 35 (2007) 5610–5624. K. Scherrer, J. Jost, Gene and genon concept: coding versus regulation. A conceptual and information-theoretic analysis of genetic storage and expression in the light of modern molecular biology, Theory Biosci. 126 (2007) 65–113. K. Scherrer, J. Jost, The gene and the genon concept: a functional and information-theoretic analysis, Mol. Syst. Biol. 3 (2007) 87. A. Taddei, G. Van Houwe, S. Nagai, I. Erb, E.J. van Nimwegen, S.M. Gasser, The functional importance of telomere clustering: global changes in gene expression result from SIR factor dispersion, Genome Res. (2009). A. Taddei, Active genes at the nuclear pore complex, Curr. Opin. Cell. Biol. 19 (2007) 305–310. M. Ruault, M. Dubarry, A. Taddei, Re-positioning genes to the nuclear envelope in mammalian cells: impact on transcription, Trends Genet. 24 (2008) 574–581. H. Hagege, P. Klous, C. Braem, E. Splinter, J. Dekker, G. Cathala, W. de Laat, T. Forne, Quantitative analysis of chromosome conformation capture assays (3C-qPCR), Nat. Protoc. 2 (2007) 1722–1733. C. Braem, B. Recolin, R.C. Rancourt, C. Angiolini, P. Barthes, P. Branchu, F. Court, G. Cathala, A.C. Ferguson-Smith, T. Forne, Genomic matrix attachment region and chromosome conformation capture quantitative real time PCR assays identify novel putative regulatory elements at the imprinted Dlk1/Gtl2 locus, J. Biol. Chem. 283 (2008) 18612–18620. F.C. Fang, S. Rimsky, New insights into transcriptional regulation by H-NS, Curr. Opin. Microbiol. 11 (2008) 113–120. E. Bouffartigues, M. Buckle, C. Badaut, A. Travers, S. Rimsky, H-NS cooperative binding to high-affinity sites in a regulatory element results in transcriptional silencing, Nat. Struct. Mol. Biol. 14 (2007) 441–448. P. Volkel, P.O. Angrand, The control of histone lysine methylation in epigenetic regulations, Biochimie 89 (2007) 1–20. C. Lavelle, A. Prunell, Chromatin polymorphism and the nucleosome superfamily: a genealogy, Cell Cycle 6 (2007) 2113–2119. C. Lavelle, Forces and torques in the nucleus: chromatin under mechanical constraints, Biochem. Cell. Biol. 87 (2009) 307–322. D. Revaud, J. Mozziconacci, L. Sabatier, C. Desmaze, C. Lavelle, Sequencedriven telomeric chromatin structure, Cell Cycle 8 (2009) 1099–1100. P. Dupaigne, C. Lavelle, A. Justome, S. Lafosse, G. Mirambeau, M. Lipinski, O. Pietrement, E. Le Cam, Rad51 polymerization reveals a new chromatin remodeling mechanism, PLoS ONE 3 (2008) e3643. F. Bertoni, M. Ponzoni, The cellular origin of mantle cell lymphoma, Int. J. Biochem. Cell. Biol. 39 (2007) 1747–1753.

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55] [56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

F. Lecointe, C. Serena, M. Velten, A. Costes, S. McGovern, J.C. Meile, J. Errington, S.D. Ehrlich, P. Noirot, P. Polard, Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks, EMBO J. 26 (2007) 4239–4251. S. Rousseaux, N. Reynoird, E. Escoffier, J. Thevenon, C. Caron, S. Khochbin, Epigenetic reprogramming of the male genome during gametogenesis and in the zygote, Reprod. Biomed. Online 16 (2008) 492–503. J. Govin, E. Escoffier, S. Rousseaux, L. Kuhn, M. Ferro, J. Thevenon, R. Catena, I. Davidson, J. Garin, S. Khochbin, C. Caron, Pericentric heterochromatin reprogramming by new histone variants during mouse spermiogenesis, J. Cell. Biol. 176 (2007) 283–294. B.T. Ruotolo, K. Giles, I. Campuzano, A.M. Sandercock, R.H. Bateman, C.V. Robinson, Evidence for macromolecular protein rings in the absence of bulk water, Science 310 (2005) 1658–1661. C. Di Primo, Real time analysis of the RNAI-RNAII-Rop complex by surface plasmon resonance: from a decaying surface to a standard kinetic analysis, J. Mol. Recognit. 21 (2008) 37–45. I. Lebars, P. Legrand, A. Aime, N. Pinaud, S. Fribourg, C. Di Primo, Exploring TAR–RNA aptamer loop–loop interaction by X-ray crystallography, UV spectroscopy and surface plasmon resonance, Nucleic Acids Res. 36 (2008) 7146–7156. C. Di Primo, I. Rudloff, S. Reigadas, A.A. Arzumanov, M.J. Gait, J.J. Toulme, Systematic screening of LNA/2¢-O-methyl chimeric derivatives of a TAR RNA aptamer, FEBS Lett. 581 (2007) 771–774. A.N. Kapanidis, E. Margeat, S.O. Ho, E. Kortkhonjia, S. Weiss, R.H. Ebright, Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism, Science 314 (2006) 1144–1147. E. Margeat, A.N. Kapanidis, P. Tinnefeld, Y. Wang, J. Mukhopadhyay, R.H. Ebright, S. Weiss, Direct observation of abortive initiation and promoter escape within single immobilized transcription complexes, Biophys. J. 90 (2006) 1419–1431. B. Heddi, N. Foloppe, E. Hantz, B. Hartmann, The DNA structure responds differently to physiological concentrations of K(þ) or Na(þ), J. Mol. Biol. 368 (2007) 1403–1411. F. Paquet, F. Culard, F. Barbault, J.C. Maurizot, G. Lancelot, NMR solution structure of the archaebacterial chromosomal protein MC1 reveals a new protein fold, Biochemistry 43 (2004) 14971–14978. G. De Vuyst, S. Aci, D. Genest, F. Culard, Atypical recognition of particular DNA sequences by the archaeal chromosomal MC1 protein, Biochemistry 44 (2005) 10369–10377. L. Ponchon, F. Dardel, Recombinant RNA technology: the tRNA scaffold, Nat. Methods 4 (2007) 571–576. S. Bernacchi, S. Freisz, C. Maechling, B. Spiess, R. Marquet, P. Dumas, E. Ennifar, Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion, Nucleic Acids Res. 35 (2007) 7128–7139. S. Freisz, K. Lang, R. Micura, P. Dumas, E. Ennifar, Binding of aminoglycoside antibiotics to the duplex form of the HIV-1 genomic RNA dimerization initiation site, Angew. Chem. Int. Ed. Engl. 47 (2008) 4110–4113. S. Marzi, A.G. Myasnikov, A. Serganov, C. Ehresmann, P. Romby, M. Yusupov, B.P. Klaholz, Structured mRNAs regulate translation initiation by binding to the platform of the ribosome, Cell 130 (2007) 1019–1031. A. Simonetti, S. Marzi, A.G. Myasnikov, A. Fabbretti, M. Yusupov, C.O. Gualerzi, B.P. Klaholz, Structure of the 30S translation initiation complex, Nature 455 (2008) 416–420. W. Winkler, A. Nahvi, R.R. Breaker, Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression, Nature 419 (2002) 952–956. S. Thore, M. Leibundgut, N. Ban, Structure of the eukaryotic thiamine pyrophosphate riboswitch with its regulatory ligand, Science 312 (2006) 1208–1211. A. Laederach, R. Das, Q. Vicens, S.M. Pearlman, M. Brenowitz, D. Herschlag, R.B. Altman, Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments, Nat. Protoc. 3 (2008) 1395–1401. A. Bancaud, G. Wagner, E.S.N. Conde, C. Lavelle, H. Wong, J. Mozziconacci, M. Barbi, A. Sivolob, E. Le Cam, L. Mouawad, J.L. Viovy, J.M. Victor, A. Prunell, Nucleosome chiral transition under positive torsional stress in single chromatin fibers, Mol. Cell. 27 (2007) 135–147. J. Mozziconacci, C. Lavelle, M. Barbi, A. Lesne, J.M. Victor, A physical model for the condensation and decondensation of eukaryotic chromosomes, FEBS Lett. 580 (2006) 368–372.