Hidden modes of DNA binding by human nuclear receptors.

Human nuclear receptors (NRs) are a superfamily of ligand-responsive transcription factors that have central roles in cellular function. Their malfunction is linked to numerous diseases, and the ability to modulate their activity with synthetic ligands has yielded 16% of all FDA-approved drugs. NRs regulate distinct gene networks, however they often function from genomic sites that lack known binding motifs. Here, to annotate genomic binding sites of known and unexamined NRs more accurately, we use high-throughput SELEX to comprehensively map DNA binding site preferences of all full-length human NRs, in complex with their ligands. Furthermore, to identify non-obvious binding sites buried in DNA-protein interactomes, we develop MinSeq Find, a search algorithm based on the MinTerm concept from electrical engineering and digital systems design. The resulting MinTerm sequence set (MinSeqs) reveal a constellation of binding sites that more effectively annotate NR-binding profiles in cells. MinSeqs also unmask binding sites created or disrupted by 52,106 single-nucleotide polymorphisms associated with human diseases. By implicating druggable NRs as hidden drivers of multiple human diseases, our results not only reveal new biological roles of NRs, but they also provide a resource for drug-repurposing and precision medicine.

Flexibility of flanking DNA is a key determinant of transcription factor affinity for the core motif.

Selective gene regulation is mediated by recognition of specific DNA sequences by transcription factors (TFs). The extremely challenging task of searching out specific cognate DNA binding sites among several million putative sites within the eukaryotic genome is achieved by complex molecular recognition mechanisms. Elements of this recognition code include the core binding sequence, the flanking sequence context, and the shape and conformational flexibility of the composite binding site. To unravel the extent to which DNA flexibility modulates TF binding, in this study, we employed experimentally guided molecular dynamics simulations of ternary complex of closely related Hox heterodimers Exd-Ubx and Exd-Scr with DNA. Results demonstrate that flexibility signatures embedded in the flanking sequences impact TF binding at the cognate binding site. A DNA sequence has intrinsic shape and flexibility features. While shape features are localized, our analyses reveal that flexibility features of the flanking sequences percolate several basepairs and allosterically modulate TF binding at the core. We also show that lack of flexibility in the motif context can render the cognate site resistant to protein-induced shape changes and subsequently lower TF binding affinity. Overall, this study suggests that flexibility-guided DNA shape, and not merely the static shape, is a key unexplored component of the complex DNA-TF recognition code.

The effect of mutation in the stem of the MicroROSE thermometer on its thermosensing ability: insights from molecular dynamics simulation studies.

A large number of bacteria have been found to govern virulence and heat shock responses using temperature sensing RNAs known as RNA thermometers (RNATs). They repress translation initiation by base pairing to the Shine-Dalgarno (SD) sequence at low temperature. Increasing the temperature induces the RNA duplex to unfold and expose the SD sequence for translation. A prime example is the ROSE thermometer module known to regulate the production of the ROSE heat shock protein in Bradyrhizobium japonicum. The unfolding of a 29-nucleotide long MicroROSE RNA element which forms a critical component encompassing the SD sequence, and three mutants that differ from it by deletion of a guanine nucleotide or mutations near the SD and stem regions have been studied using high temperature molecular dynamics simulations. The simulations reveal the progressive manner in which a biologically functional RNA thermometer unfolds. Our simulations reveal that deletion of the highly conserved G10 residue, opposite to the SD region leads to the formation of a stable RNA helix that has lost its thermosensing ability. Mutations of bases A5 → U5 and U25 → A25 near the stem increase the thermosensing ability due to the allosteric effect which leads to a global destabilization effect on the structure. The temperature-dependant regulation of this thermometer has been investigated by estimation of differences in the unfolding paths by calculating individual residue fluctuation, stacking energy, the contact map plot and the lifetime dynamics plot of non-Watson-Crick hydrogen bonds at three different temperatures. Results reveal that partial unfolding at higher temperature starts from the hairpin tetra loop end and terminates at the stem region through the SD associated region. Two canonical hydrogen bonds between U9-A22 and four non-canonical hydrogen bonds between G10-G21 and U6-U24 around the internal loop play an important role in partial melting of the RNA helix. These results demonstrate how small alterations in RNA structure can regulate gene expression and illuminate the molecular basis of the function of an important bacterial regulatory motif.

RNA-mediated translation regulation in viral genomes: computational advances in the recognition of sequences and structures.

RNA structures are widely distributed across all life forms. The global conformation of these structures is defined by a variety of constituent structural units such as helices, hairpin loops, kissing-loop motifs and pseudoknots, which often behave in a modular way. Their ubiquitous distribution is associated with a variety of functions in biological processes. The location of these structures in the genomes of RNA viruses is often coordinated with specific processes in the viral life cycle, where the presence of the structure acts as a checkpoint for deciding the eventual fate of the process. These structures have been found to adopt complex conformations and exert their effects by interacting with ribosomes, multiple host translation factors and small RNA molecules like miRNA. A number of such RNA structures have also been shown to regulate translation in viruses at the level of initiation, elongation or termination. The role of various computational studies in the preliminary identification of such sequences and/or structures and subsequent functional analysis has not been fully appreciated. This review aims to summarize the processes in which viral RNA structures have been found to play an active role in translational regulation, their global conformational features and the bioinformatics/computational tools available for the identification and prediction of these structures.

Variation of gene expression in plants is influenced by gene architecture and structural properties of promoters.

In higher eukaryotes, gene architecture and structural properties of promoters have emerged as significant factors influencing variation in number of transcripts (expression level) and specificity of gene expression in a tissue (expression breadth), which eventually shape the phenotype. In this study, transcriptome data of different tissue types at various developmental stages of A. thaliana, O. sativa, S. bicolor and Z. mays have been used to understand the relationship between properties of gene components and its expression. Our findings indicate that in plants, among all gene architecture and structural properties of promoters, compactness of genes in terms of intron content is significantly linked to gene expression level and breadth, whereas in human an exactly opposite scenario is seen. In plants, for the first time we have carried out a quantitative estimation of effect of a particular trait on expression level and breadth, by using multiple regression analysis and it confirms that intron content of primary transcript (as %) is a powerful determinant of expression breadth. Similarly, further regression analysis revealed that among structural properties of the promoters, stability is negatively linked to expression breadth, while DNase1 sensitivity strongly governs gene expression breadth in monocots and gene expression level in dicots. In addition, promoter regions of tissue specific genes are found to be enriched with TATA box and Y-patch motifs. Finally, multi copy orthologous genes in plants are found to be longer, highly regulated and tissue specific.

Flexibility and structure of flanking DNA impact transcription factor affinity for its core motif.

Spatial and temporal expression of genes is essential for maintaining phenotype integrity. Transcription factors (TFs) modulate expression patterns by binding to specific DNA sequences in the genome. Along with the core binding motif, the flanking sequence context can play a role in DNA-TF recognition. Here, we employ high-throughput in vitro and in silico analyses to understand the influence of sequences flanking the cognate sites in binding of three most prevalent eukaryotic TF families (zinc finger, homeodomain and bZIP). In vitro binding preferences of each TF toward the entire DNA sequence space were correlated with a wide range of DNA structural parameters, including DNA flexibility. Results demonstrate that conformational plasticity of flanking regions modulates binding affinity of certain TF families. DNA duplex stability and minor groove width also play an important role in DNA-TF recognition but differ in how exactly they influence the binding in each specific case. Our analyses further reveal that the structural features of preferred flanking sequences are not universal, as similar DNA-binding folds can employ distinct DNA recognition modes.

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.

Dynamics of physiologically relevant noncanonical DNA structures: an overview from experimental and theoretical studies.

DNA is a complex molecule with phenomenal inherent plasticity and the ability to form different hydrogen bonding patterns of varying stabilities. These properties enable DNA to attain a variety of structural and conformational polymorphic forms. Structurally, DNA can exist in single-stranded form or as higher-order structures, which include the canonical double helix as well as the noncanonical duplex, triplex and quadruplex species. Each of these structural forms in turn encompasses an ensemble of dynamically heterogeneous conformers depending on the sequence composition and environmental context. In vivo, the widely populated canonical B-DNA attains these noncanonical polymorphs during important cellular processes. While several investigations have focused on the structure of these noncanonical DNA, studying their dynamics has remained nontrivial. Here, we outline findings from some recent advanced experimental and molecular simulation techniques that have significantly contributed toward understanding the complex dynamics of physiologically relevant noncanonical forms of DNA.

Identification of putative promoters in 48 eukaryotic genomes on the basis of DNA free energy.

Transcription is an intricate mechanism and is orchestrated at the promoter region. The cognate motifs in the promoters are observed in only a subset of total genes across different domains of life. Hence, sequence-motif based promoter prediction may not be a holistic approach for whole genomes. Conversely, the DNA structural property, duplex stability is a characteristic of promoters and can be used to delineate them from other genomic sequences. In this study, we have used a DNA duplex stability based algorithm 'PromPredict' for promoter prediction in a broad range of eukaryotes, representing various species of yeast, worm, fly, fish, and mammal. Efficiency of the software has been tested in promoter regions of 48 eukaryotic systems. PromPredict achieves recall values, which range from 68 to 92% in various eukaryotes. PromPredict performs well in mammals, although their core promoter regions are GC rich. 'PromPredict' has also been tested for its ability to predict promoter regions for various transcript classes (coding and non-coding), TATA-containing and TATA-less promoters as well as on promoter sequences belonging to different gene expression variability categories. The results support the idea that differential DNA duplex stability is a potential predictor of promoter regions in various genomes.

DNA structural features of eukaryotic TATA-containing and TATA-less promoters.

Eukaryotic genes can be broadly classified as TATA-containing and TATA-less based on the presence of TATA box in their promoters. Experiments on both classes of genes have revealed a disparity in the regulation of gene expression and cellular functions between the two classes. In this study, we report characteristic differences in promoter sequences and associated structural properties of the two categories of genes in six different eukaryotes. We have analyzed three structural features, DNA duplex stability, bendability, and curvature along with the distribution of A-tracts, G-quadruplex motifs, and CpG islands. The structural feature analyses reveal that while the two classes of gene promoters are distinctly different from each other, the properties are also distinguishable across the six organisms.

RNAHelix: computational modeling of nucleic acid structures with Watson-Crick and non-canonical base pairs.

Comprehensive analyses of structural features of non-canonical base pairs within a nucleic acid double helix are limited by the availability of a small number of three dimensional structures. Therefore, a procedure for model building of double helices containing any given nucleotide sequence and base pairing information, either canonical or non-canonical, is seriously needed. Here we describe a program RNAHelix, which is an updated version of our widely used software, NUCGEN. The program can regenerate duplexes using the dinucleotide step and base pair orientation parameters for a given double helical DNA or RNA sequence with defined Watson-Crick or non-Watson-Crick base pairs. The original structure and the corresponding regenerated structure of double helices were found to be very close, as indicated by the small RMSD values between positions of the corresponding atoms. Structures of several usual and unusual double helices have been regenerated and compared with their original structures in terms of base pair RMSD, torsion angles and electrostatic potentials and very high agreements have been noted. RNAHelix can also be used to generate a structure with a sequence completely different from an experimentally determined one or to introduce single to multiple mutation, but with the same set of parameters and hence can also be an important tool in homology modeling and study of mutation induced structural changes.

Unveiling DNA structural features of promoters associated with various types of TSSs in prokaryotic transcriptomes and their role in gene expression.

Next-generation sequencing studies have revealed that a variety of transcripts are present in the prokaryotic transcriptome and a significant fraction of them are functional, being involved in various regulatory activities apart from coding for proteins. Identification of promoters associated with different transcripts is necessary for characterization of the transcriptome. Promoter regions have been shown to have unique structural features as compared with their flanking region, in organisms covering all domains of life. Here we report an in silico analysis of DNA sequence dependent structural properties like stability, bendability and curvature in the promoter region of six different prokaryotic transcriptomes. Using these structural features, we predicted promoters associated with different categories of transcripts (mRNA, internal, antisense and non-coding), which constitute the transcriptome. Promoter annotation using structural features is fairly accurate and reliable with about 50% of the primary promoters being characterized by all three structural properties while at least one property identifies 95%. We also studied the relative differences of these structural features in terms of gene expression and found that the features, viz. lower stability, lesser bendability and higher curvature are more prominent in the promoter regions which are associated with high gene expression as compared with low expression genes. Hence, promoters, which are associated with higher gene expression, get annotated well using DNA structural features as compared with those, which are linked to lower gene expression.

Data on diverse roles of helix perturbations in membrane proteins.

The various structural variations observed in TM helices of membrane proteins have been deconstructed into 9 distinct types of helix perturbations. These perturbations are defined by the deviation of TM helices from the predominantly observed linear α-helical conformation, to form 310- and π-helices, as well as adopting curved and kinked geometries. The data presented here supplements the article 'Helix perturbations in Membrane Proteins Assist in Inter-helical Interactions and Optimal Helix Positioning in the Bilayer' (A. Shelar, M. Bansal, 2016) [1]. This data provides strong evidence for the role of various helix perturbations in influencing backbone torsion angles of helices, mediating inter-helical interactions, oligomer formation and accommodation of hydrophobic residues within the bilayer. The methodology used for creation of various datasets of membrane protein families (Sodium/Calcium exchanger and Heme Copper Oxidase) has also been mentioned.

The role of sequence in altering the unfolding pathway of an RNA pseudoknot: a steered molecular dynamics study.

Mechanical unfolding studies on Ribonucleic Acid (RNA) structures are a subject of tremendous interest as they shed light on the principles of higher order assembly of these structures. Pseudoknotting is one of the most elementary ways in which this higher order assembly is achieved as discrete secondary structural units in RNA are brought in close proximity to form a tertiary structure. Using steered molecular dynamics (SMD) simulations, we have studied the unfolding of five RNA pseudoknot structures that differ from each other either by base substitutions in helices or loops. Our SMD simulations reveal the manner in which a biologically functional RNA pseudoknot unfolds and the effect of changes in the primary structure on this unfolding pathway, providing necessary insights into the driving forces behind the functioning of these structures. We observed that an A → C mutation in the loop sequence makes the pseudoknot far more resistant against force induced disruption relative to its wild type structure. In contrast to this, a base-pair substitution GC → AU near the pseudoknot junction region renders it more vulnerable to this disruption. The quantitative estimation of differences in the unfolding paths was carried out using force extension curves, potential of mean force profiles, and the opening of different Watson-Crick and non-Watson-Crick interactions. The results provide a quantified view in which the unfolding paths of the small RNA structures can be used for investigating the programmability of RNA chains for designing RNA switches and aptamers as their biological folding and unfolding could be assessed and manipulated.

Structural features of DNA are conserved in the promoter region of orthologous genes across different strains of Helicobacter pylori.

Promoter regions play a key role in the process of transcription initiation and gene expression, hence promoter identification is an inherent component of the genome annotation process. Identification and characterization of promoters in fully sequenced genomes is a challenging and complex task. An analysis of sequence-dependent DNA structural properties in the promoter region of orthologous and non-orthologous genes can help in characterizing promoters and also provide insights into transcription initiation. Various structural properties, such as duplex stability, protein-induced bendability and intrinsic curvature of promoter sequences have been calculated and compared for 10 different strains of Helicobacter pylori genomes, and it is found that promoter regions in orthologous and non-orthologous genes show distinct trends for these properties, with orthologous genes showing sharper low-stability peak, lower bendability and higher curvature. The average GC content of orthologous genes is higher than that of non-orthologous genes, and relative stability-based promoter annotation tool PromPredict performs better for orthologous genes than non-orthologous genes. The characteristic sequence-dependent structural properties of promoters show significant differences between orthologous and non-orthologous genes. Interestingly, these structural properties of promoters are conserved, but the genes themselves vary in their evolutionary selection rate.

Structural and functional analyses of PolyProline-II helices in globular proteins.

PolyProline-II (PPII) helices are defined as a continuous stretch of a protein chain in which the constituent residues have backbone torsion angle (φ, ψ) values of (-75°, 145°) and take up an extended left handed helical conformation, without any intra-chain hydrogen bonds. They are found to occur quite frequently in protein structures, with their number exceeding that of π-helices, though it is considerably less than that of α-helices and ß-strands. A relatively new procedure, ASSP, for the identification of regular secondary structures using Cα trace identifies 3597 PPII-helices in 3582 protein chains, solved at resolution ⩽2.0Å. Taking advantage of this significantly expanded database of PPII-helices, we have analyzed their structural and functional roles as well as determined the amino acid propensity within and around them. Though Pro residues are highly preferred, their presence is not a mandatory requirement for the formation of PPII-helices, since ∼40% PPII-helices were found to contain no Pro residues. Aromatic amino acids are avoided within this helix, while Gly, Asn and Asp residues are preferred in the proximal flanking regions. The PPII-helices range from 3 to 13 residues in length with the average twist and rise being -121.2°±9.2° and 3.0ű0.1Å respectively. A majority (∼72%) of PPII-helices were found to occur in conjunction with α-helices and ß-strands, and serve as linkers as well. The analysis of various intra-helical non-bonded interactions revealed frequent presence of CH⋯O H-bonds. PPII-helices participate in maintaining the three-dimensional structure of proteins and are important constituents of binding motifs involved in various biological functions.

Helix perturbations in membrane proteins assist in inter-helical interactions and optimal helix positioning in the bilayer.

Transmembrane (TM) helices in integral membrane proteins are primarily α-helical in structure. Here we analyze 1134 TM helices in 90 high resolution membrane proteins and find that apart from the widely prevalent α-helices, TM regions also contain stretches of 310 (3 to 8 residues) and π-helices (5 to 19 residues) with distinct sequence signatures. The various helix perturbations in TM regions comprise of helices with kinked geometry, as well as those with an interspersed 310/π-helical fragment and show high occurrence in a few membrane proteins. Proline is frequently present at sites of these perturbations, but it is neither a necessary nor a sufficient requirement. Helix perturbations are also conserved within a family of membrane proteins despite low sequence identity in the perturbed region. Furthermore, a perturbation influences the geometry of the TM helix, mediates inter-helical interactions within and across protein chains and avoids hydrophobic mismatch of the helix termini with the bilayer. An analysis of π-helices in the TM regions of the heme copper oxidase superfamily shows that interspersed π-helices can vary in length from 6 to 19 amino acids or be entirely absent, depending upon the protein function. The results presented here would be helpful for prediction of 310 and π-helices in TM regions and can assist the computational design of membrane proteins.

Genome-Wide Targets Regulated by the OsMADS1 Transcription Factor Reveals Its DNA Recognition Properties.

OsMADS1 controls rice (Oryza sativa) floral fate and organ development. Yet, its genome-wide targets and the mechanisms underlying its role as a transcription regulator controlling developmental gene expression are unknown. We identify 3112 gene-associated OsMADS1-bound sites in the floret genome. These occur in the vicinity of transcription start sites, within gene bodies, and in intergenic regions. Majority of the bound DNA contained CArG motif variants or, in several cases, only A-tracts. Sequences flanking the binding peak had a higher AT nucleotide content, implying that broader DNA structural features may define in planta binding. Sequences for binding by other transcription factor families like MYC, AP2/ERF, bZIP, etc. are enriched in OsMADS1-bound DNAs. Target genes implicated in transcription, chromatin remodeling, cellular processes, and hormone metabolism were enriched. Combining expression data from OsMADS1 knockdown florets with these DNA binding data, a snapshot of a gene regulatory network was deduced where targets, such as AP2/ERF and bHLH transcription factors and chromatin remodelers form nodes. We show that the expression status of these nodal factors can be altered by inducing the OsMADS1-GR fusion protein and present a model for a regulatory cascade where the direct targets of OsMADS1, OsbHLH108/SPT, OsERF034, and OsHSF24, in turn control genes such as OsMADS32 and OsYABBY5 This cascade, with other similar relationships, cumulatively contributes to floral organ development. Overall, OsMADS1 binds to several regulatory genes and, probably in combination with other factors, controls a gene regulatory network that ensures rice floret development.

Conformational stabilities of iminoallantoin and its base pairs in DNA: implications for mutagenicity.

The types of mutations induced by oxidatively damaged products of DNA are continuously in debate. For example, some biochemical studies have proposed that guanidinohydantoin (Gh) would induce exclusively G to C mutations, while other studies have predicted a mixture of various mutations including G to C, G to T and G to A. In addition to the nature of mutations, the exact reasons of these mutations are also not properly understood. It is suggested that Gh can easily isomerize to iminoallantoin (Ia) in a pH-dependent manner and the transition becomes complete at pH > 8. In order to understand Gh/Ia-induced mutations, we have here studied the role of the most stable tautomer of Ia in the R- and S-enantiomeric configurations in promoting mismatch base pair complexes in DNA by employing a density functional theoretical (DFT) approach. It is found that Ia can have 39 different possible tautomeric forms each in the R- and S-enantiomeric configurations, out of which the most stable tautomer would involve the deprotonation of the N1 atom and protonation of the N3 atom. The most stable tautomer of Ia can adopt three different rotameric conformations (Ia1, Ia2, and Ia3) of comparable stabilities. It is further revealed that these rotamers of Ia can interact with different bases of DNA in 88 different possible ways. However, the interaction of G with Ia3 in both the anti- and syn-conformations would be the most stable. It is further revealed that the base pairing patterns, binding energies and electronic environments of anti-Ia3:G and G:T complexes are similar. In addition to this, it is also found that the binding patterns and energies of Gh1:G and Ia3:G complexes are similar. Based on these results, it is proposed that under physiological conditions, Gh1 may be responsible for the observed G to C mutations in DNA, while in an acidic environment Ia3 may be responsible for the same mutations. This study has led to a solid foundation for further high resolution structural studies to completely unravel Ia-induced mutagenicity in DNA.

Insights into the Structural Dynamics of Nucleocytoplasmic Transport of tRNA by Exportin-t.

Exportin-t (Xpot) transports mature 5'- and 3'-end processed tRNA from the nucleus to the cytoplasm by associating with a small G-protein Ran (RAs-related nuclear protein), in the nucleus. The release of tRNA in cytoplasm involves RanGTP hydrolysis. Despite the availability of crystal structures of nuclear and cytosolic forms of Xpot, the molecular details regarding the sequential events leading to tRNA release and subsequent conformational changes occurring in Xpot remain unknown. We have performed a combination of classical all-atom and accelerated molecular dynamics simulations on a set of complexes involving Xpot to study a range of features including conformational flexibility of free and cargo-bound Xpot and functionally critical contacts between Xpot and its cargo. The systems investigated include free Xpot and its different complexes, bound either to Ran (GTP/GDP) or tRNA or both. This approach provided a statistically reliable estimate of structural dynamics of Xpot after cargo release. The mechanistic basis for Xpot opening after cargo release has been explained in terms of dynamic structural hinges, about which neighboring region could be displaced to facilitate the nuclear to cytosolic state transition. Post-RanGTP hydrolysis, a cascade of events including local conformational change in RanGTP and loss of critical contacts at Xpot/tRNA interface suggest factors responsible for eventual release of tRNA. The level of flexibility in different Xpot complexes varied depending on the arrangement of individual HEAT repeats. Current study provides one of the most comprehensive and robust analysis carried out on this protein using molecular dynamics schemes.