Living systems are capable on the one hand of eliciting a coordinated response to changing environments (also known as adaptation), and on the other hand, they are capable of reproducing themselves. Notably, adaptation to environmental change requires the monitoring of the surroundings, while reproduction requires monitoring oneself. These two tasks appear separate and make use of different sources of information. Yet, both the process of adaptation as well as that of reproduction are inextricably coupled to alterations in genomic DNA expression, while a cell behaves as an indivisible unity in which apparently independent processes and mechanisms are both integrated and coordinated. We argue that at the most basic level, this integration is enabled by the unique property of the DNA to act as a double coding device harboring two logically distinct types of information. We review biological systems of different complexities and infer that the inter-conversion of these two distinct types of DNA information represents a fundamental self-referential device underlying both systemic integration and coordinated adaptive responses.

Broadening of signals from atoms at interfaces can often be a limiting factor in applying solution NMR to the structure determination of complexes. Common contributors to such problems include exchange between free and bound states and the increased molecular weight of complexes relative to the free components, but another cause that can be more difficult to deal with occurs when conformational dynamics within the interface takes place at an intermediate rate on the chemical shift timescale. In this work we show how a carefully chosen mutation in the protein HMG-D rescued such a situation, making possible high-resolution structure determination of its complex with a dA2 bulge DNA ligand designed to mimic a natural DNA bend, and thereby revealing a new spatial organization of the complex.

Ce chapitre discute des preuves de l'évolution du code génétique vers sa forme actuelle. Il suggère que la sélection initiale des paires codon-anticodon dépendait de la conservation d'une structure d'ARN relativement stable contenant l'anticodon et également de la stabilisation des structures d'ARNm simple brin par empilement de bases.

The temporal evolution of the genetic code has long been a topic of controversial discussion, yet the origins of the present-day code are crucial to our understanding of not only the code itself, but also to the evolution of proteins and how they are able to do the work necessary to maintain biological homeostasis. A primary question in considerations of the evolution of the genetic code is how both the accuracy and stability of the crucial codon–anticodon recognition step can be ensured. On this view, evolution of both the codon–anticodon interaction and also of protein function, and hence of the mix of utilized amino acids, depends on relative structural stability, which facilitates the overall efficiency of the chemical processes with respect to the cumulative rates of chemical reactions involved. Discussions initially centered on the triplet nature of the code, and then on the relative thermal stability of the necessary codon–anticodon interactions in an RNA world.

In this article we describe the bacterial growth cycle as a closed, self-reproducing, or autopoietic circuit, reestablishing the physiological state of stationary cells initially inoculated in the growth medium. In batch culture, this process of self-reproduction is associated with the gradual decline in available metabolic energy and corresponding change in the physiological state of the population as a function of "travelled distance" along the autopoietic path. We argue that this directional alteration of cell physiology is both reflected in and supported by sequential gene expression along the chromosomal OriC-Ter axis. We propose that during the E. coli growth cycle, the spatiotemporal order of gene expression is established by coupling the temporal gradient of supercoiling energy to the spatial gradient of DNA thermodynamic stability along the chromosomal OriC-Ter axis.

The coordination of bacterial genomic transcription involves an intricate network of interdependent genes encoding nucleoid-associated proteins (NAPs), DNA topoisomerases, RNA polymerase subunits and modulators of transcription machinery. The central element of this homeostatic regulatory system, integrating the information on cellular physiological state and producing a corresponding transcriptional response, is the multi-subunit RNA polymerase (RNAP) holoenzyme. In this review article, we argue that recent observations revealing DNA topoisomerases and metabolic enzymes associated with RNAP supramolecular complex support the notion of structural coupling between transcription machinery, DNA topology and cellular metabolism as a fundamental device coordinating the spatiotemporal genomic transcription. We analyse the impacts of various combinations of RNAP holoenzymes and global transcriptional regulators such as abundant NAPs, on genomic transcription from this viewpoint, monitoring the spatiotemporal patterns of couplons-overlapping subsets of the regulons of NAPs and RNAP sigma factors. We show that the temporal expression of regulons is by and large, correlated with that of cognate regulatory genes, whereas both the spatial organization and temporal expression of couplons is distinctly impacted by the regulons of NAPs and sigma factors. We propose that the coordination of the growth phase-dependent concentration gradients of global regulators with chromosome configurational dynamics determines the spatiotemporal patterns of genomic expression.

During a long and extremely productive scientific life Michael Waring made substantial contributions to the understanding of certain fundamental physical properties of the DNA double helix. In this review I summarize his early studies on DNA supercoiling and his later conclusions on the role of natural DNA base analogues in determining DNA stiffness and nucleosome positioning. The latter are reassessed in the context of subsequent advances in the understanding of the parameters affecting base-step deformability. Michael's scientific career spanned nearly six extremely productive decades. He started out soon after the double-helical structure of DNA was proposed by Watson and Crick, in 1953. And from then on DNA was always the main focus of his research, with an especial interest in the mechanism of how DNA-binding drugs interacted with the genetic material. In this retrospective on Michael's science I shall concentrate on his contributions to the understanding of DNA structure and function rather than his voluminous exploration of drug-DNA interactions. The latter, I'm sure, will be discussed by those more intimately involved in those experiments. I first became aware of Michael's science in the mid-1960s when I was working as a research student at the Medical Research Council Laboratory of Molecular Biology (MRC-LMB) in Cambridge, England. I was studying the initiation of transcription on polyoma DNA which had very kindly been provided by Lionel Crawford (then at the Institute of virology in Glasgow, Scotland) and I saw that Michael was scheduled to present a Tea Club in the University of Cambridge Department of Biochemistry on the interaction of ethidium bromide with polyoma DNA. Michael had newly returned from a post-doctoral stint at the Carnegie Institute in Washington DC, USA in the group of Roy Britten and had taken up a University Demonstratorship in the Department of Biochemistry, resuming his pre-doctoral interest in antimicrobial agents that bind DNA. At the Tea Club Michael recounted the successful outcome of the confluence of his and Lionel's research interests. Ethidium bromide is a phenanthridine trypanocide which forms a complex with double-stranded DNA.[1, 2] By analogy with Leonard Lerman's work on the mode of acridine binding to DNA,[3] Michael, together with Watson Fuller at King's College (University of London, England), had proposed that the planar ring system of ethidium bromide intercalated between the base pairs of the DNA, causing a local unwinding of the double helix.[4] At that time polyoma DNA was of significant interest. The molecule is circular and was the source of the discovery that DNA can be supercoiled.[5-7] DNA supercoiling results from an excess or deficiency of turns in the double helix relative to the number of turns in a “relaxed” unconstrained DNA molecule. The evidence at the time suggested that supercoiled polyoma DNA had a deficiency of turns.[7, 8] An experimental test of this hypothesis would be to systematically alter the number of turns in the double helix by the binding of ethidium bromide and ask whether the drug affected a proxy manifestation of superhelicity—the hydrodynamic properties of the DNA molecule as determined by analytical ultracentrifugation. The results showed that as the drug binding to DNA initially increased, the intercalation into the molecules first caused a loss of supercoiling and then with a further increase in drug binding a superhelical configuration was regained (Figure 1).[9] Knowing that ethidium bromide intercalation untwists the double helix, the initial loss of supercoiling at low levels of drug binding showed that the supercoiling in the untreated DNA must be induced by a deficiency of right-handed double helical turns, causing the molecule to take up a conformation in which the duplex was wound about itself in a right-handed sense; a form termed a negative supercoil.[8] On this interpretation higher levels of drug binding introduced superhelical turns of the opposite sense—positive supercoiling—resulting in the recovery of superhelicity. From the ratio of drug to DNA at the point where supercoiling disappeared, it was possible to calculate the number of superhelical turns in a polyoma DNA molecule. To do this required knowledge of the ethidium bromide unwinding angle. In Michael and Lionel's study, relying on the inferences of a previous modeling study, with Watson Fuller, of the ethidium bromide interaction with DNA this was taken to be 12° to 13°.[4] Their work indicated the untreated molecule of polyoma virus DNA contained at least 12 supercoiling turns. It was the first direct demonstration that superhelicity involved a change in the winding of the DNA double helix. Lionel and Michael then followed up the study by determining the number of superhelical turns in the circular DNA molecules of a papilloma virus, another type of DNA virus, to be approximately 20.[10] The genomes of papilloma viruses are in general ~60% bigger than those of polyoma virus and the result implied that the superhelical density (no. of superhelical turns/100 bp DNA) was approximately the same for both types of virus. Subsequent studies quantitatively refined these conclusions. Later experimental studies using different techniques showed that the ethidium bromide unwinding angle is ~23° to 24° rather than the 12° to 13° initially assumed.[11] Consequently the number of superhelical turns in polyoma and papilloma DNA molecules is likely approximately twice as large as the initial determinations. By a fortunate coincidence, 40 years later Michael had the opportunity to return to supercoiled DNA. In collaboration with Robert Henderson in the Department of Biochemistry at Cambridge, he added atomic form microscopy (AFM) to his toolkit. AFM enabled them to directly visualize how echinomycin, a bis-intercalating DNA binding drug, affected DNA superhelicity. As drug binding to the DNA increased they observed the transitions from a negative supercoil first to an open circle and then to a positive supercoil.[12] From the late-1960s to the mid-1980s Michael's work concentrated almost exclusively on drug-DNA interactions. And then Michael—always ready to embrace new technologies—was introduced by Horace Drew, working next door as a post-doc in Aaron Klug's group in the MRC-LMB to “DNA footprinting.” The new technique utilized the high-resolution gel electrophoresis methods, developed in parallel with DNA sequencing methodology established during that period, to identify precisely the location of ligand binding sites in defined DNA sequences by virtue of their protection from enzymatic or chemical attack. Starting with his favorite drug, echinomycin, Michael, first with Keith Fox (his Jesus College undergraduate Part II supervisee, then Ph.D. student and subsequently post-doc, and now at the University of Southampton, UK) and afterwards with many collaborators, used this technique extensively—as also did Peter Dervan (Caltech, USA) and others studying drug-DNA interactions—to obtain detailed insights into the binding properties, binding sites and DNA-sequence preferences of a range of DNA-binding drugs with a variety of binding modes.[13] One of the first fruits of this approach was the determination of the sequence-specific binding sites of two anthracycline drugs, doxorubicin and daunomycin.[14] These studies confirmed Michael's earlier experiments suggesting that, like echinomycin, these drugs acted via DNA intercalation.[15] A further crucial general insight that emerged, initially from Michael's work on echinomycin with post-doc Christian Bailly, was the importance of the purine 2-amino group as a critical sequence-specific recognition element for drug binding to both CG-containing and AT-containing sites. To demonstrate this unequivocally, the guanosine residues in an echinomycin binding site were replaced with inosine. (Strictly the base is hypoxanthine and the nucleoside inosine and for consistency the base pair should be designated H-C and not I-C. Nevertheless, since Michael and other workers always used the term inosine, its use is retained here.) Inosine lacks the 2-amino group present in guanosine and the substitution practically abolished echinomycin binding.[16] This crucial experiment opened up a new field in Michael's research. He realized that substitution with base analogues in DNA might provide important insights into the physical properties of DNA, in particular its flexibility. Horace Drew (MRC-LMB) had also introduced Michael to chromatin and in 1986 they had inferred that both echinomycin and distamycin (a non-intercalating minor-groove binding antibiotic) caused rotation of the DNA duplex with respect to the surface of the octamer in nucleosome cores, thus modulating octamer positioning.[17] Subsequent studies with his postdoc José Portugal showed that other minor-groove binding drugs, including netropsin and berenil, had the same effect but the interaction of echinomycin with the nucleosome was more complex.[18] Octamer-bound DNA is tightly bent therefore Michael proposed to utilize the histone octamer as well as other DNA-bending proteins as proxies for determining the relative flexibility of analogue-substituted DNA sequences. The strategy was to substitute bases in natural sequences with analogues that altered the pattern of the exocyclic groups in the minor and major grooves of DNA, and to investigate how these substitutions affected its physical properties. The chosen substitutions were inosine (I) and diaminopurine (DAP) which respectively remove and add a 2-amino group in the minor groove, and uracil (U) and 5-methyl cytosine (M), which respectively remove and add a methyl group in the major groove. All these base analogues occur naturally. M is a well-characterized epigenetic marker while I, DAP and U all separately occur as replacements for the normal bases in the genomic DNA of certain bacteriophages and cyanophages (summarized in Crippen et al.[19]). In a series of publications[12, 20-25] this approach demonstrated that the presence of exocyclic groups, such as 2-amino purine in the minor groove and methyl groups in the major groove, decreased affinity for the histone octamer, so that the DNA sequences with the highest affinity for the octamer completely lacked exocyclic groups (the uracil and inosine substitution). Conversely those in which all purines contained a 2-amino group and all 5-pyrimidine positions were methylated, (the diaminopurine and 5-methylcytosine substitution) had the lowest affinity (Figure 2).[20] A very similar result was obtained using simple DNA-bending proteins HMG-D and FIS.[21, 22] The inference was that the presence of exocyclic groups decreased DNA flexibility (otherwise expressed as an increase in DNA rigidity). Histone octamer affinity is an indirect measure of DNA flexibility. Fortunately an opportunity arose for a more direct determination after a colleague asked Michael whether he could study the properties of a set of natural high-affinity octamer-binding sequences.[26] With these sequences in hand Michael and Robert Henderson used AFM to determine whether the single I or DAP substitutions altered the persistence length of DNA fragments.[23] The results largely paralleled octamer binding experiments with the same sequences. I decreased and DAP increased the persistence length while increasing and decreasing, respectively, the affinity of the DNA for the histone octamer (Figure 3).[23] The increased stiffness of DAP-substituted DNA revealed by AFM is substantial and was subsequently confirmed in other studies.[11, 27, 28] Not only did the base substitutions modulate the affinity for the histone octamer, but DNA footprinting demonstrated that they, like distamycin, also changed the rotational orientation of the DNA sequence on the surface of the histone octamer. In particular, DAP substitution in some, but not all, of the sequences tested switched the rotational phase so that the orientations of the major and minor grooves relative to the octamer surface were reversed compared to the unsubstituted sequences. M substitution had a similar effect, but only with one of the tested seqences.[19] Conversely I substitution enhanced a pre-existing rotational preference in several sequences.[19, 23] There was one further intriguing finding from the experiments. Both the DAP singly substituted and the DAP+I doubly substituted molecules were always more compact than a normal B-DNA molecule with the same number of base pairs. In other words, the helical rise was reduced. Additionally, the magnitude of the reduction was sequence dependent. The average helical rise for B-DNA is ~3.45 Å/bp while that for A-DNA is ~2.56 Å/bp.[29] Values of 2.9-3.1, 3.16, 3.1 and 2.79 Å/bp have been reported for DAP substituted DNA sequences[12, 23, 27, 28] together with one of 2.52 Å/bp for DAP+I substituted DNA.[12] Inosine substitution alone also results in a slight reduction in the helical rise.[12, 23] The results of the base-substitution experiments raised many pertinent questions. What was the structure of the DAP-substituted sequences with a reduced helical rise? How did DAP increase DNA stiffness? Why did certain substitutions, and not others, alter the rotational positioning of nucleosomal DNA? Michael always wanted to follow up on these questions but unfortunately, as so often, circumstances intervened and the experiments never got done. With hindsight and more available knowledge some of the answers have become clearer therefore in the following paragraphs I shall attempt to provide a coherent interpretation of the experimental results and a perspective on the legacy of Michael's experiments. The original experimental data contained two important clues to the nature of the conformational change induced by DAP. One came from the DNase cleavage patterns of the substituted DNAs on the surface of the histone octamer. It is known that DNase I binds across the minor groove of DNA and cuts each strand independently. For classical B-form DNA this results in an average stagger between the upper and lower strands of about 3 nucleotides in the 3′ direction. This distance corresponds to a perpendicular line drawn between the two strands across the minor groove of the DNA and depends on the inclination of the base pairs to the helical axis such that the stagger increases as the inclination becomes more negative.[30, 31] The data for the nucleosome-bound DNA showed that while the stagger for unsubstituted DNA was 3-4 bp (and thus the DNA was likely B-form), the values for I substituted and DAP substituted DNA were respectively 2 bp and 5 bp.[23] The second clue was the observation that both the I and DAP substitutions separately reduced the CD maximum at ~275 Å characteristic of B-DNA. However, whereas I substitution, in three out of four examples shifted the maximum to a shorter wavelength (~260 Å), DAP substitution shifted it to a longer wavelength (~295 Å).[23] This shift in the CD maximum from I to DAP substituted DNA thus paralleled the shift in the cleavage stagger and hence in the inferred inclination of the base pairs to the helical axis. Variations in the inclination angle and CD spectra are also observed in different DNA conformers. For the classical DNA structures the inclination in A-DNA is on average +20°, in B-DNA 0° and in C- and D-DNA more negative (−8° in C-DNA fibers; −16° in the closely related D-DNA).[29] The transition to C-DNA is also associated with a shift in the CD maximum to longer wavelengths,[32] consistent with the opposite chiralities of the local helical axis in A-DNA and C-DNA. The same shift to longer wavelengths in in the CD spectrum maximum is also observed accompanying the DAP-induced transition to the X-form of poly(dAT).[33] The inference is that DAP substitution favors a DNA duplex with characteristics in common with C-DNA, although not necessarily conforming to a classical C-DNA structure. In this model the reduction in helical rise in DAP substituted DNA would be associated with an increased negative inclination angle in just the same way that the shorter helical rise in A-DNA is associated with an increased positive inclination angle. Overall the data imply that DAP substitution increases the helical twist with an associated change in the inclination angle while I substitution has the opposite effect. However, by itself a structural shift to a C-like DNA form does not fully explain the observed changes in DNA flexibility. The data shows that while the CD spectrum of the doubly substituted sequence (I + DAP) may be indicative of a different DNA conformation it does not correlate with the persistence length proxy of histone octamer affinity.[23] Sequences doubly substituted with DAP and I have very similar CD spectra but their affinity for the octamer is intermediate between that of the respective single substituted sequences (the ID substitution in Figure 2).[18, 23] In other words although the effect of DAP on DNA conformation is dominant to that of I, its effect on flexibility is not. This is consistent with the observation that the effect of DAP substitution on uranyl-mediated cleavage of double stranded DNA is very similar to that of I + DAP substitution but completely different from that of I substitution.[24] While DAP substitution reduces cleavage in A/T rich regions it enhances cleavage in G-C rich regions.[24] Both findings are suggestive of DAP inducing a concerted conformational switch in the helical architecture. These observations address the fundamental nature of DNA flexibility. Direct measurements of persistence length and of affinity for the histone octamer average the properties of a DNA molecule as a whole. These averages approximate to the sum of the deformabilities of individual base-steps. But how to explain the effects of DNA-base substitutions on flexibility in this context? Not only do the base substitutions likely affect the organization of both bound water molecules and cations, they also affect the number of hydrogen bonds involved in base pairing and the stacking interactions between adjacent base pairs. The alteration in the number of hydrogen bonds has two general consequences. The DNA melting temperature is raised in the DAP substituted sequences in which all base pairs contain three H-bonds and correspondingly lowered in the I substituted sequences.[23, 34] Additionally loss of the third pairing H-bond from G-C pairs on substitution with I would allow a greater freedom for propeller twisting. Indeed when substituted in a dA-dT tract where the A-T pairs have a high propeller twist,[34] I-C base pairs can confer intrinsic curvature.[35] On these considerations alone I substituted DNA would be expected to be more flexible and more unwound with a wider minor groove than the unsubstituted DNA. The explanation for the increased rigidity of DAP-substituted DNA is less obvious. A priori it might be anticipated that the increased occupancy of the minor groove by 2-amino groups would be favored by a wider minor groove. However, an alternative, and not necessarily mutually exclusive explanation, is that the double helical structure adjusts to the increased 2-amino group occupancy by overtwisting to minimize steric clashes, especially in sequences with adjacent purine residues.[27] Such a shift would likely confer rigidity by restricting the range of local conformations that the DNA base pairs could assume. Ultimately changes in DNA groove width and the transitions between DNA conformers are coupled to changes in the geometry of the sugar-phosphate backbone. Variation in this geometry, which is particularly prominent in DNA molecules bent around the histone octamer, is thus linked to base-step deformability. Arguably the base pair substitutions influence the balance of the transitions between different backbone conformations.[36-42] Should a substitution strongly favor a particular backbone geometry the deformability of an affected base-step would be restricted translating to an increase in global stiffness.[36, 40, 41] Conversely any change facilitating transitions between alternative backbone geometries would enhance flexibility. One such switch is between two phosphate conformations, termed BI and BII.[36, 37] The relative proportions of the these conformations in B-DNA crystal structures are correlated with groove width.[42] The BII conformation is more favored in C-DNA,[43] and also in octamer-bound DNA in positions where the minor groove points in towards the histone octamer.[36, 41] The inference is that DAP substitution favors the BII configuration while I substitution not only favors the BI configuration—albeit to a lesser extent—but also enhances the transitions between the BI and BII configurations thereby increasing the available range of base-step geometries available to the substituted base-steps. In contrast to DAP, M substitution is believed to strongly favor the BI configuration.[44] Perhaps then it is no surprise that of the variants tested, the doubly substituted D + M DNA had the lowest affinity for the histone octamer.[20] With reduced deformability it was, in the parlance of the field, “frustrated.”[45] One speculation in this context is that the removal of the 5-methyl group of thymine (the U substitution) might also enhance the probability of the BI/BII transition. These considerations are directly relevant to the problem of nucleosome positioning. Balsubramanian et al. calculated that DNA wrapped on the surface of the octamer adopted a more C-like conformation at an inward-facing minor groove and a more A-like conformation at an outward-facing minor groove.[46] This finding is completely consistent with the distribution of the BI and BII geometries in nucleosome crystal structures.[41] Based on these arguments a major factor influencing nucleosome positioning on a DNA sequence is the relative deformability of its component base-steps. The enhancement (to ±1 bp) of the precision of positioning of all but one of the tested sequences by I substitution suggests that I substitutions minimize local (position-dependent) impediments to optimal base-step deformability. The one exception was a sequence for which positioning was dependent on runs of less deformable and phased (dA)(dT) tracts conferring intrinsic curvature. These would obviously not be directly affected by I substitution. In the examples where substitutions altered the rotational positions, decreased deformability of particular base-steps could change the relative preferred orientations of different sequence elements. For example, DAP substitution would confer extra rigidity on the A/T-rich sequences which then would be less able to accommodate to the curvature required for an inward-facing minor groove. The observation that distamycin and other minor-groove binding drugs reverse the rotational orientation of octamer-bound DNA[17, 18] could be explained in a similar manner assuming that the drug locally alters the rigidity of A/T-rich sequences. Taken as a whole, the above considerations amply justify the answer to a more profound question posed by Buttinelli et al.—“Why then should A-T and G-C base pairs be apparently preferred by natural selection?”[20] They suggested that the distribution of exocyclic groups combined with base-stacking interactions in canonical DNA could modulate groove dimensions and “together these characteristics would allow bendability [aka flexibility or deformability] to be combined with specificity.” Although science was a dominating feature of his life, Michael was a man of very diverse interests. He was a connoisseur of wine and music, and especially enjoyed playing the College organ. He also had a passion for flying, a pursuit that was an everlasting topic of anecdotal recollections amongst his friends. Perhaps my own experience was typical. We were cruising at 3000′ over a wide estuary when Michael said to me “Could you take over for a few minutes while I go behind. It's very simple.” Never mind that this was the first time I'd ever ventured into a small plane let alone flown one. Apart from my tendency to put the nose up a little he was absolutely right and the experience turned out to be much less arduous than the occasion when I operated the regulator of an old-fashioned steam tank engine trundling along a rural branch line in the West of England. One other enduring memory was from yet another drug-related meeting, this time in Brno, a city renowned for its teaching of organ music. Michael had heard that a local church housed a well-tuned instrument. So he went to locate it and ushered us into the building. After quite a long wait the music sounded around the nave. I shall never know how Michael persuaded the guardians of the organ to let him play but the effect was not only truly memorable—it was typical of the man. I am most grateful to Jean Thomas for help with the article. The authors declare no competing interests. Data sharing is not applicable to this article as no new data were created or analyzed in this study.

In this article, we summarize our current understanding of the bacterial genetic regulation brought about by decades of studies using the Escherichia coli model. It became increasingly evident that the cellular genetic regulation system is organizationally closed, and a major challenge is to describe its circular operation in quantitative terms. We argue that integration of the DNA analog information (i.e., the probability distribution of the thermodynamic stability of base steps) and digital information (i.e., the probability distribution of unique triplets) in the genome provides a key to understanding the organizational logic of genetic control. During bacterial growth and adaptation, this integration is mediated by changes of DNA supercoiling contingent on environmentally induced shifts in intracellular ionic strength and energy charge. More specifically, coupling of dynamic alterations of the local intrinsic helical repeat in the structurally heterogeneous DNA polymer with structural-compositional changes of RNA polymerase holoenzyme emerges as a fundamental organizational principle of the genetic regulation system. We present a model of genetic regulation integrating the genomic pattern of DNA thermodynamic stability with the gene order and function along the chromosomal OriC-Ter axis, which acts as a principal coordinate system organizing the regulatory interactions in the genome.

Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.

We have investigated the structure of the most compact 30-nm chromatin fibres by modelling those with 2-start or 1-start crossed-linker organisations. Using an iterative procedure we obtained possible structural solutions for fibres of the highest possible compaction permitted by physical constraints, including the helical repeat of linker DNA. We find that this procedure predicts a quantized nucleosome repeat length (NRL) and that only fibres with longer NRLs (≥197 bp) can more likely adopt the 1-start organisation. The transition from 2-start to 1-start fibres is consistent with reported differing binding modes of the linker histone. We also calculate that in 1-start fibres the DNA constrains more torsion (as writhe) than 2-start fibres with the same NRL and that the maximum constraint obtained is in accord with previous experimental results. We posit that the coiling of the fibre is driven by overtwisting of linker DNA which, in the most compact forms - for example, in echinoderm sperm and avian erythrocytes - could adopt a helical repeat of ∼10 bp/turn. We argue that in vivo the total twist of linker DNA could be modulated by interaction with other abundant chromatin-associated proteins and by epigenetic modifications of the C-terminal tail of linker histones.

Disordered proteins play an essential role in a wide variety of biological processes, and are often posttranslationally modified. One such protein is histone H1; its highly disordered C-terminal tail (CH1) condenses internucleosomal linker DNA in chromatin in a way that is still poorly understood. Moreover, CH1 is phosphorylated in a cell cycle-dependent manner that correlates with changes in the chromatin condensation level. Here we present a model system that recapitulates key aspects of the in vivo process, and also allows a detailed structural and biophysical analysis of the stages before and after condensation. CH1 remains disordered in the DNA-bound state, despite its nanomolar affinity. Phase-separated droplets (coacervates) form, containing higher-order assemblies of CH1/DNA complexes. Phosphorylation at three serine residues, spaced along the length of the tail, has little effect on the local properties of the condensate. However, it dramatically alters higher-order structure in the coacervate and reduces partitioning to the coacervate phase. These observations show that disordered proteins can bind tightly to DNA without a disorder-to-order transition. Importantly, they also provide mechanistic insights into how higher-order structures can be exquisitely sensitive to perturbation by posttranslational modifications, thus broadening the repertoire of mechanisms that might regulate chromatin and other macromolecular assemblies.

The structure of compact 30‐nm chromatin fibres is still debated. We present here a novel unified model that reconciles all experimental observations into a single framework. We propose that compact fibres are formed by the interdigitation of the two nucleosome stacks in a 2‐start crossed‐linker structure to form a single stack. This process requires that the dyad orientation of successive nucleosomes relative to the helical axis alternates. The model predicts that, as observed experimentally, the fibre‐packing density should increase in a stepwise manner with increasing linker length. This model structure can also incorporate linker DNA of varying lengths.

We describe a biophysical approach that enables changes in the structure of DNA to be followed during nucleosome formation in in vitro reconstitution with either the canonical “Widom” sequence or a judiciously mutated sequence. The rapid non-perturbing photochemical analysis presented here provides ‘snapshots’ of the DNA configuration at any given moment in time during nucleosome formation under a very broad range of reaction conditions. Changes in DNA photochemical reactivity upon protein binding are interpreted as being mainly induced by alterations in individual base pair roll angles. The results strengthen the importance of the role of an initial (H3/H4)2 histone tetramer-DNA interaction and highlight the modulation of this early event by the DNA sequence. (H3/H4)2 binding precedes and dictates subsequent H2A/H2B-DNA interactions, which are less affected by the DNA sequence, leading to the final octameric nucleosome. Overall, our results provide a novel, exciting way to investigate those biophysical properties of DNA that constitute a crucial component in nucleosome formation and stabilization.

We argue that dynamic changes in DNA supercoiling in vivo determine both how DNA is packaged and how it is accessed for transcription and for other manipulations such as recombination. In both bacteria and eukaryotes, the principal generators of DNA superhelicity are DNA translocases, supplemented in bacteria by DNA gyrase. By generating gradients of superhelicity upstream and downstream of their site of activity, translocases enable the differential binding of proteins which preferentially interact with respectively more untwisted or more writhed DNA. Such preferences enable, in principle, the sequential binding of different classes of protein and so constitute an essential driver of chromatin organization.

The bacterial gene regulatory regions often demonstrate distinctly organized arrays of RNA polymerase binding sites of ill-defined function. Previously we observed a module of closely spaced polymerase binding sites upstream of the canonical promoter of the Escherichia coli fis operon. FIS is an abundant nucleoid-associated protein involved in adjusting the chromosomal DNA topology to changing cellular physiology. Here we show that simultaneous binding of the polymerase at the canonical fis promoter and an upstream transcriptionally inactive site stabilizes a RNAP oligomeric complex in vitro. We further show that modulation of the upstream binding of RNA polymerase affects the fis promoter activity both in vivo and in vitro. The effect of the upstream RNA polymerase binding on the fis promoter activity depends on the spatial arrangement of polymerase binding sites and DNA supercoiling. Our data suggest that a specific DNA geometry of the nucleoprotein complex stabilized on concomitant binding of RNA polymerase molecules at the fis promoter and the upstream region acts as a topological device regulating the fis transcription. We propose that transcriptionally inactive RNA polymerase molecules can act as accessory factors regulating the transcription initiation from a nearby promoter.

The proposal of a double-helical structure for DNA over 60 years ago provided an eminently satisfying explanation for the heritability of genetic information. But why is DNA, and not RNA, now the dominant biological information store? We argue that, in addition to its coding function, the ability of DNA, unlike RNA, to adopt a B-DNA structure confers advantages both for information accessibility and for packaging. The information encoded by DNA is both digital - the precise base specifying, for example, amino acid sequences - and analogue. The latter determines the sequence-dependent physicochemical properties of DNA, for example, its stiffness and susceptibility to strand separation. Most importantly, DNA chirality enables the formation of supercoiling under torsional stress. We review recent evidence suggesting that DNA supercoiling, particularly that generated by DNA translocases, is a major driver of gene regulation and patterns of chromosomal gene organization, and in its guise as a promoter of DNA packaging enables DNA to act as an energy store to facilitate the passage of translocating enzymes such as RNA polymerase.

Recent studies strongly suggest that in bacterial cells the order of genes along the chromosomal origin-to-terminus axis is determinative for regulation of the growth phase-dependent gene expression. The prediction from this observation is that positional displacement of pleiotropic genes will affect the genetic regulation and hence, the cellular phenotype. To test this prediction we inserted the origin-proximal dusB-fis operon encoding the global regulator FIS in the vicinity of replication terminus on both arms of the Escherichia coli chromosome. We found that the lower fis gene dosage in the strains with terminus-proximal dusB-fis operons was compensated by increased fis expression such that the intracellular concentration of FIS was homeostatically adjusted. Nevertheless, despite unchanged FIS levels the positional displacement of dusB-fis impaired the competitive growth fitness of cells and altered the state of the overarching network regulating DNA topology, as well as the cellular response to environmental stress, hazardous substances and antibiotics. Our finding that the chromosomal repositioning of a regulatory gene can determine the cellular phenotype unveils an important yet unexplored facet of the genetic control mechanisms and paves the way for novel approaches to manipulate bacterial physiology.

In this article, we sketch out a holistic methodology used for exploring how the genetic program is encoded in a 2-D genetic map of a bacterial chromosome. We argue that the major problem resides in the conceptual integration of the two logically distinct types of information encoded in the chiral double-helical DNA polymer. This integration is accomplished by mapping the genetic function on the genomic sequence organisation and therefore is potentially applicable to any chromosome. The vast generalisation achieved by this approach necessarily ignores exquisite details, yet it is fundamental in providing comprehensive methodology for exploring the role of the DNA sequence organisation in harnessing genetic information and sustaining biological order.