Designed architectural proteins that tune DNA looping in bacteria.

Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

Insights into DNA solvation found in protein-DNA structures.

The proteins that bind double-helical DNA present various microenvironments that sense and/or induce signals in the genetic material. The high-resolution structures of protein-DNA complexes reveal the nature of both the microenvironments and the conformational responses in DNA and protein. Complex networks of interactions within the structures somehow tie the protein and DNA together and induce the observed spatial forms. Here we show how the cumulative buildup of amino acid atoms around the sugars, phosphates, and bases in different protein-DNA complexes produces a binding cloud around the double helix and how different types of atoms fill that cloud. Rather than focusing on the principles of molecular binding and recognition suggested by the arrangements of amino acids and nucleotides in the macromolecular complexes, we consider the proteins in contact with DNA as organized solvents. We describe differences in the mix of atoms that come in closest contact with DNA, subtle sequence-dependent features in the microenvironment of the sugar-phosphate backbone, a direct link between the localized buildup of ionic species and the electrostatic potential surfaces of the DNA bases, and sites of atomic buildup above and below the basepair planes that transmit the unique features of the base environments along the chain backbone. The inferences about solvation that can be drawn from the survey provide new stimuli for improvement of nucleic acid force fields and fresh ideas for exploration of the properties of DNA in solution.

Web 3DNA 2.0 for the analysis, visualization, and modeling of 3D nucleic acid structures.

Web 3DNA (w3DNA) 2.0 is a significantly enhanced version of the widely used w3DNA server for the analysis, visualization, and modeling of 3D nucleic-acid-containing structures. Since its initial release in 2009, the w3DNA server has continuously served the community by making commonly-used features of the 3DNA suite of command-line programs readily accessible. However, due to the lack of updates, w3DNA has clearly shown its age in terms of modern web technologies and it has long lagged behind further developments of 3DNA per se. The w3DNA 2.0 server presented here overcomes all known shortcomings of w3DNA while maintaining its battle-tested characteristics. Technically, w3DNA 2.0 implements a simple and intuitive interface (with sensible defaults) for increased usability, and it complies with HTML5 web standards for broad accessibility. Featurewise, w3DNA 2.0 employs the most recent version of 3DNA, enhanced with many new functionalities, including: the automatic handling of modified nucleotides; a set of 'simple' base-pair and step parameters for qualitative characterization of non-Watson-Crick double-helical structures; new structural parameters that integrate the rigid base plane and the backbone phosphate group, the two nucleic acid components most reliably determined with X-ray crystallography; in silico base mutations that preserve the backbone geometry; and a notably improved module for building models of single-stranded RNA, double-helical DNA, Pauling triplex, G-quadruplex, or DNA structures 'decorated' with proteins. The w3DNA 2.0 server is freely available, without registration, at http://web.x3dna.org.

Effects of Noncanonical Base Pairing on RNA Folding: Structural Context and Spatial Arrangements of G·A Pairs.

Noncanonical base pairs play important roles in assembling the three-dimensional structures critical to the diverse functions of RNA. These associations contribute to the looped segments that intersperse the canonical double-helical elements within folded, globular RNA molecules. They stitch together various structural elements, serve as recognition elements for other molecules, and act as sites of intrinsic stiffness or deformability. This work takes advantage of new software (DSSR) designed to streamline the analysis and annotation of RNA three-dimensional structures. The multiscale structural information gathered for individual molecules, combined with the growing number of unique, well-resolved RNA structures, makes it possible to examine the collective features deeply and to uncover previously unrecognized patterns of chain organization. Here we focus on a subset of noncanonical base pairs involving guanine and adenine and the links between their modes of association, secondary structural context, and contributions to tertiary folding. The rigorous descriptions of base-pair geometry that we employ facilitate characterization of recurrent geometric motifs and the structural settings in which these arrangements occur. Moreover, the numerical parameters hint at the natural motions of the interacting bases and the pathways likely to connect different spatial forms. We draw attention to higher-order multiplexes involving two or more G·A pairs and the roles these associations appear to play in bridging different secondary structural units. The collective data reveal pairing propensities in base organization, secondary structural context, and deformability and serve as a starting point for further multiscale investigations and/or simulations of RNA folding.

Nucleosome-free DNA regions differentially affect distant communication in chromatin.

Communication between distantly spaced genomic regions is one of the key features of gene regulation in eukaryotes. Chromatin per se can stimulate efficient enhancer-promoter communication (EPC); however, the role of chromatin structure and dynamics in this process remains poorly understood. Here we show that nucleosome spacing and the presence of nucleosome-free DNA regions can modulate chromatin structure/dynamics and, in turn, affect the rate of EPC in vitro and in silico. Increasing the length of internucleosomal linker DNA from 25 to 60 bp results in more efficient EPC. The presence of longer nucleosome-free DNA regions can positively or negatively affect the rate of EPC, depending upon the length and location of the DNA region within the chromatin fiber. Thus the presence of histone-free DNA regions can differentially affect the efficiency of EPC, suggesting that gene regulation over a distance could be modulated by changes in the length of internucleosomal DNA spacers.

Toward a Universal Structural and Energetic Model for Prokaryotic Promoters.

With almost no consensus promoter sequence in prokaryotes, recruitment of RNA polymerase (RNAP) to precise transcriptional start sites (TSSs) has remained an unsolved puzzle. Uncovering the underlying mechanism is critical for understanding the principle of gene regulation. We attempted to search the hidden code in ∼16,500 promoters of 12 prokaryotes representing two kingdoms in their structure and energetics. Twenty-eight fundamental parameters of DNA structure including backbone angles, basepair axis, and interbasepair and intrabasepair parameters were used, and information was extracted from x-ray crystallography data. Three parameters (solvation energy, hydrogen-bond energy, and stacking energy) were selected for creating energetics profiles using in-house programs. DNA of promoter regions was found to be inherently designed to undergo a change in every parameter undertaken for the study, in all prokaryotes. The change starts from some distance upstream of TSSs and continues past some distance from TSS, hence giving a signature state to promoter regions. These signature states might be the universal hidden codes recognized by RNAP. This observation was reiterated when randomly selected promoter sequences (with little sequence conservation) were subjected to structure generation; all developed into very similar three-dimensional structures quite distinct from those of conventional B-DNA and coding sequences. Fine structural details at important motifs (viz. -11, -35, and -75 positions relative to TSS) of promoters reveal novel to our knowledge and pointed insights for RNAP interaction at these locations; it could be correlated with how some particular structural changes at the -11 region may allow insertion of RNAP amino acids in interbasepair space as well as facilitate the flipping out of bases from the DNA duplex.

Contributions of Sequence to the Higher-Order Structures of DNA.

One of the critical unanswered questions in genome biophysics is how the primary sequence of DNA bases influences the global properties of very-long-chain molecules. The local sequence-dependent features of DNA found in high-resolution structures introduce irregularities in the disposition of adjacent residues that facilitate the specific binding of proteins and modulate the global folding and interactions of double helices with hundreds of basepairs. These features also determine the positions of nucleosomes on DNA and the lengths of the interspersed DNA linkers. Like the patterns of basepair association within DNA, the arrangements of nucleosomes in chromatin modulate the properties of longer polymers. The intrachromosomal loops detected in genomic studies contain hundreds of nucleosomes, and given that the simulated configurations of chromatin depend on the lengths of linker DNA, the formation of these loops may reflect sequence-dependent information encoded within the positioning of the nucleosomes. With knowledge of the positions of nucleosomes on a given genome, methods are now at hand to estimate the looping propensities of chromatin in terms of the spacing of nucleosomes and to make a direct connection between the DNA base sequence and larger-scale chromatin folding.

DSSR: an integrated software tool for dissecting the spatial structure of RNA.

Insight into the three-dimensional architecture of RNA is essential for understanding its cellular functions. However, even the classic transfer RNA structure contains features that are overlooked by existing bioinformatics tools. Here we present DSSR (Dissecting the Spatial Structure of RNA), an integrated and automated tool for analyzing and annotating RNA tertiary structures. The software identifies canonical and noncanonical base pairs, including those with modified nucleotides, in any tautomeric or protonation state. DSSR detects higher-order coplanar base associations, termed multiplets. It finds arrays of stacked pairs, classifies them by base-pair identity and backbone connectivity, and distinguishes a stem of covalently connected canonical pairs from a helix of stacked pairs of arbitrary type/linkage. DSSR identifies coaxial stacking of multiple stems within a single helix and lists isolated canonical pairs that lie outside of a stem. The program characterizes 'closed' loops of various types (hairpin, bulge, internal, and junction loops) and pseudoknots of arbitrary complexity. Notably, DSSR employs isolated pairs and the ends of stems, whether pseudoknotted or not, to define junction loops. This new, inclusive definition provides a novel perspective on the spatial organization of RNA. Tests on all nucleic acid structures in the Protein Data Bank confirm the efficiency and robustness of the software, and applications to representative RNA molecules illustrate its unique features. DSSR and related materials are freely available at http://x3dna.org/.

DNA topology confers sequence specificity to nonspecific architectural proteins.

Topological constraints placed on short fragments of DNA change the disorder found in chain molecules randomly decorated by nonspecific, architectural proteins into tightly organized 3D structures. The bacterial heat-unstable (HU) protein builds up, counter to expectations, in greater quantities and at particular sites along simulated DNA minicircles and loops. Moreover, the placement of HU along loops with the "wild-type" spacing found in the Escherichia coli lactose (lac) and galactose (gal) operons precludes access to key recognition elements on DNA. The HU protein introduces a unique spatial pathway in the DNA upon closure. The many ways in which the protein induces nearly the same closed circular configuration point to the statistical advantage of its nonspecificity. The rotational settings imposed on DNA by the repressor proteins, by contrast, introduce sequential specificity in HU placement, with the nonspecific protein accumulating at particular loci on the constrained duplex. Thus, an architectural protein with no discernible DNA sequence-recognizing features becomes site-specific and potentially assumes a functional role upon loop formation. The locations of HU on the closed DNA reflect long-range mechanical correlations. The protein responds to DNA shape and deformability­the stiff, naturally straight double-helical structure­rather than to the unique features of the constituent base pairs. The structures of the simulated loops suggest that HU architecture, like nucleosomal architecture, which modulates the ability of regulatory proteins to recognize their binding sites in the context of chromatin, may influence repressor-operator interactions in the context of the bacterial nucleoid.

Arginine-phosphate salt bridges between histones and DNA: intermolecular actuators that control nucleosome architecture.

Structural bioinformatics and van der Waals density functional theory are combined to investigate the mechanochemical impact of a major class of histone-DNA interactions, namely, the formation of salt bridges between arginine residues in histones and phosphate groups on the DNA backbone. Principal component analysis reveals that the configurational fluctuations of the sugar-phosphate backbone display sequence-specific directionality and variability, and clustering of nucleosome crystal structures identifies two major salt-bridge configurations: a monodentate form in which the arginine end-group guanidinium only forms one hydrogen bond with the phosphate, and a bidentate form in which it forms two. Density functional theory calculations highlight that the combination of sequence, denticity, and salt-bridge positioning enables the histones to apply a tunable mechanochemical stress to the DNA via precise and specific activation of backbone deformations. The results suggest that selection for specific placements of van der Waals contacts, with high-precision control of the spatial distribution of intermolecular forces, may serve as an underlying evolutionary design principle for the structure and function of nucleosomes, a conjecture that is corroborated by previous experimental studies.

What controls DNA looping?

The looping of DNA provides a means of communication between sequentially distant genomic sites that operate in tandem to express, copy, and repair the information encoded in the DNA base sequence. The short loops implicated in the expression of bacterial genes suggest that molecular factors other than the naturally stiff double helix are involved in bringing the interacting sites into close spatial proximity. New computational techniques that take direct account of the three-dimensional structures and fluctuations of protein and DNA allow us to examine the likely means of enhancing such communication. Here, we describe the application of these approaches to the looping of a 92 base-pair DNA segment between the headpieces of the tetrameric Escherichia coli Lac repressor protein. The distortions of the double helix induced by a second protein--the nonspecific nucleoid protein HU--increase the computed likelihood of looping by several orders of magnitude over that of DNA alone. Large-scale deformations of the repressor, sequence-dependent features in the DNA loop, and deformability of the DNA operators also enhance looping, although to lesser degrees. The correspondence between the predicted looping propensities and the ease of looping derived from gene-expression and single-molecule measurements lends credence to the derived structural picture.

Biophysical Reviews: a transition in the journal.

This Editorial for Volume 16 Issue 2 first describes the issue contents before describing some upcoming events within Biophysical Reviews and concludies with an announcement on the transition of Chief Editors thanks to the outgoing Chief Editor.

Biophysical reviews Special issue call: The 21st IUPAB Congress 2024 Kyoto Japan.

This commentary describes an open call for submissions to the upcoming Biophysical Reviews' Special Issue: The 21st IUPAB Congress 2024 Kyoto Japan. The submission deadline is July 1st of 2024. Interested parties are requested to make contact with the Special Issue editors prior to submission.

Internucleosomal interactions mediated by histone tails allow distant communication in chromatin.

Action across long distances on chromatin is a hallmark of eukaryotic transcriptional regulation. Although chromatin structure per se can support long-range interactions, the mechanisms of efficient communication between widely spaced DNA modules in chromatin remain a mystery. The molecular simulations described herein suggest that transient binary internucleosomal interactions can mediate distant communication in chromatin. Electrostatic interactions between the N-terminal tails of the core histones and DNA enhance the computed probability of juxtaposition of sites that lie far apart along the DNA sequence. Experimental analysis of the rates of communication in chromatin constructs confirms that long-distance communication occurs efficiently and independently of distance on tail-containing, but not on tailless, chromatin. Taken together, our data suggest that internucleosomal interactions involving the histone tails are essential for highly efficient, long-range communication between regulatory elements and their targets in eukaryotic genomes.

Structural insights into the role of architectural proteins in DNA looping deduced from computer simulations.

Bacterial gene expression is regulated by DNA elements that often lie far apart along the genomic sequence, but come close together during genetic processing. The intervening residues form loops, which are organized by the binding of various proteins. For example, the Escherichia coli Lac repressor protein binds DNA operators, separated by 92 or 401 bp, and suppresses the formation of gene products involved in the metabolism of lactose. The system also includes several highly abundant architectural proteins, such as the histone-like (heat-unstable) HU protein, which severely deform the double helix upon binding. In order to gain a better understanding of how the naturally stiff DNA double helix forms the short loops detected in vivo, we have developed new computational methods to study the effects of various non-specific binding proteins on the three-dimensional configurational properties of DNA sequences. The present article surveys the approach that we use to generate ensembles of spatially constrained protein-decorated DNA structures (minicircles and Lac repressor-mediated loops) and presents some of the insights gained from the correspondence between computation and experiment about the potential contributions of architectural and regulatory proteins to DNA looping and gene expression.

Weak operator binding enhances simulated Lac repressor-mediated DNA looping.

The 50th anniversary of Biopolymers coincides closely with the like celebration of the discovery of the Escherichia coli (lac) lactose operon, a classic genetic system long used to illustrate the influence of biomolecular structure on function. The looping of DNA induced by the binding of the Lac repressor protein to sequentially distant operator sites on DNA continues to serve as a paradigm for understanding long-range genomic communication. Advances in analyses of DNA structures and in incorporation of proteins in computer simulations of DNA looping allow us to address long-standing questions about the role of protein-mediated DNA loop formation in transcriptional control. Here we report insights gained from studies of the sequence-dependent contributions of the natural lac operators to Lac repressor-mediated DNA looping. Novel superposition of the ensembles of protein-bound operator structures derived from NMR measurements reveals variations in DNA folding missed in conventional structural alignments. The changes in folding affect the predicted ease with which the repressor induces loop formation and the ways that DNA closes between the protein headpieces. The peeling of the auxiliary operators away from the repressor enhances the formation of loops with the 92-bp wildtype spacing and hints of a structural reason behind their weak binding.

The RNA backbone plays a crucial role in mediating the intrinsic stability of the GpU dinucleotide platform and the GpUpA/GpA miniduplex.

The side-by-side interactions of nucleobases contribute to the organization of RNA, forming the planar building blocks of helices and mediating chain folding. Dinucleotide platforms, formed by side-by-side pairing of adjacent bases, frequently anchor helices against loops. Surprisingly, GpU steps account for over half of the dinucleotide platforms observed in RNA-containing structures. Why GpU should stand out from other dinucleotides in this respect is not clear from the single well-characterized H-bond found between the guanine N2 and the uracil O4 groups. Here, we describe how an RNA-specific H-bond between O2'(G) and O2P(U) adds to the stability of the GpU platform. Moreover, we show how this pair of oxygen atoms forms an out-of-plane backbone 'edge' that is specifically recognized by a non-adjacent guanine in over 90% of the cases, leading to the formation of an asymmetric miniduplex consisting of 'complementary' GpUpA and GpA subunits. Together, these five nucleotides constitute the conserved core of the well-known loop-E motif. The backbone-mediated intrinsic stabilities of the GpU dinucleotide platform and the GpUpA/GpA miniduplex plausibly underlie observed evolutionary constraints on base identity. We propose that they may also provide a reason for the extreme conservation of GpU observed at most 5'-splice sites.

Long-range effects of histone point mutations on DNA remodeling revealed from computational analyses of SIN-mutant nucleosome structures.

The packaging of DNA into nucleosomes impedes the binding and access of molecules involved in its processing. The SWI/SNF multi-protein assembly, found in yeast, is one of many regulatory factors that stimulate the remodeling of DNA required for its transcription. Amino-acid point mutations in histones H3 or H4 partially bypass the requirement of the SWI/SNF complex in this system. The mechanisms underlying the observed remodeling, however, are difficult to discern from the crystal structures of nucleosomes bearing these so-called SIN (SWI/SNF INdependent) mutations. Here, we report detailed analyses of the conformations and interactions of the histones and DNA in these assemblies. We find that the loss of direct protein-DNA contacts near point-mutation sites, reported previously, is coupled to unexpected additional long-range effects, i.e. loss of intermolecular contacts and accompanying DNA conformational changes at sequentially and spatially distant sites. The SIN mutations seemingly transmit information relevant to DNA binding across the nucleosome. The energetic cost of deforming the DNA to the states found in the SIN-mutant structures helps to distinguish the mutants that show phenotypes in yeast from those that do not. Models incorporating these deformed dimer steps suggest ways that nucleosomal DNA may be remodeled during its biological processing.

3DNALandscapes: a database for exploring the conformational features of DNA.

3DNALandscapes, located at: http://3DNAscapes.rutgers.edu, is a new database for exploring the conformational features of DNA. In contrast to most structural databases, which archive the Cartesian coordinates and/or derived parameters and images for individual structures, 3DNALandscapes enables searches of conformational information across multiple structures. The database contains a wide variety of structural parameters and molecular images, computed with the 3DNA software package and known to be useful for characterizing and understanding the sequence-dependent spatial arrangements of the DNA sugar-phosphate backbone, sugar-base side groups, base pairs, base-pair steps, groove structure, etc. The data comprise all DNA-containing structures--both free and bound to proteins, drugs and other ligands--currently available in the Protein Data Bank. The web interface allows the user to link, report, plot and analyze this information from numerous perspectives and thereby gain insight into DNA conformation, deformability and interactions in different sequence and structural contexts. The data accumulated from known, well-resolved DNA structures can serve as useful benchmarks for the analysis and simulation of new structures. The collective data can also help to understand how DNA deforms in response to proteins and other molecules and undergoes conformational rearrangements.

emDNA - A Tool for Modeling Protein-decorated DNA Loops and Minicircles at the Base-pair Step Level.

Computational modeling of nucleic acids plays an important role in molecular biology, enhancing our general understanding of the relationship between structure and function. Biophysical studies have provided a wealth of information on how double-helical DNA responds to proteins and other molecules in its local environment but far less understanding of the larger scale structural responses found in protein-decorated loops and minicircles. Current computational models of DNA range from detailed all-atom molecular dynamics studies, which produce rich time and spatially dependent depictions of small DNA fragments, to coarse-grained simulations, which sacrifice detailed physical and chemical information to treat larger-scale systems. The treatment of DNA used here, at the base-pair step level with rigid-body parameters, allows one to develop models hundreds of base pairs long from local, sequence-specific features found from experiment. The emDNA software takes advantage of this framework, producing optimized structures of DNA at thermal equilibrium with built-in or user-generated elastic models. The program, in combination with the case studies included in this article, allows users of any skill level to develop and investigate mesoscale models of their own design. The functionality of emDNA includes a tool to incorporate experiment-specific configurations, e.g., protein-bound and/or melted DNA from known high-resolution structures, within higher-order 3D models by fixing the orientation and position of user-specified base pairs. The software provides a new avenue into multiscale genetic modeling, giving a wide range of users a deeper understanding of DNA mesoscale organization and the opportunity to pose new questions in genetic research. The publicly available emDNA software, including build instructions and usage information, is available on GitHub (https://nicocvn.github.io/emDNA/).