Backbone NMR assignment of the nucleotide binding domain of the Bacillus subtilis ABC multidrug transporter BmrA in the post-hydrolysis state

ATP binding cassette (ABC) proteins are present in all phyla of life and form one of the largest protein families. The Bacillus subtilis ABC transporter BmrA is a functional homodimer that can extrude many different harmful compounds out of the cell. Each BmrA monomer is composed of a transmembrane domain (TMD) and a nucleotide binding domain (NBD). While the TMDs of ABC transporters are sequentially diverse, the highly conserved NBDs harbor distinctive conserved motifs that enable nucleotide binding and hydrolysis, interdomain communication and that mark a protein as a member of the ABC superfamily. In the catalytic cycle of an ABC transporter, the NBDs function as the molecular motor that fuels substrate translocation across the membrane via the TMDs and are thus pivotal for the entire transport process. For a better understanding of the structural and dynamic consequences of nucleotide interactions within the NBD at atomic resolution, we determined the 1H, 13C and 15N backbone chemical shift assignments of the 259 amino acid wildtype BmrA-NBD in its post-hydrolytic, ADP-bound state.

Mutation rate variations in the human genome are encoded in DNA shape

Single nucleotide mutation rates have critical implications for human evolution and genetic diseases. Accurate modeling of these mutation rates has long remained an open problem since the rates vary substantially across the human genome. A recent model, however, explained much of the variation by considering higher order nucleotide interactions in the local (7-mer) sequence context around mutated nucleotides. Despite this model’s predictive value, we still lack a clear understanding of the biophysical mechanisms underlying the variations in genome-wide mutation rates. DNA shape features are geometric measurements of DNA structural properties, such as helical twist and tilt, and are known to capture information on interactions between neighboring nucleotides within a local context. Motivated by this characteristic of DNA shape features, we used them to model mutation rates in the human genome. These DNA shape feature based models improved both the accuracy (up to 14%) and the interpretability over the current nucleotide sequence-based models. The models also discovered the specific shape features that capture the most variability in mutation rates, and distinguished between the most and the least mutated sequence contexts, thus characterizing mutation promoting properties of the genomic DNA. To our knowledge, this is the first attempt that demonstrates the structural underpinnings of nucleotide mutations in the human genome and lays the groundwork for future studies to incorporate DNA shape information in modeling genetic variations.

Rewiring the specificity of extracytoplasmic function sigma factors

Bacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators’ DNA binding and precise determination of biologically significant binding sites. Here we address these challenges by solving the DNA-specificity code of extracytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their putative regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the autoregulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid–nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic footprinting pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that some ECF σs are conserved global regulators controlling many genes throughout the genome, which are important under many conditions, while others are local regulators, controlling a few closely linked genes in response to specific stimuli in select species. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

Rewiring the specificity of extra-cytoplasmic function sigma factors

SUMMARYBacterial genomes are being sequenced at an exponentially increasing rate, but our inability to decipher their transcriptional wiring limits our ability to derive new biology from these sequences. De novo determination of regulatory interactions requires accurate prediction of regulators’ DNA binding and precise determination of biologically significant binding sites. Here, we address these challenges by solving the DNA-specificity code of extra-cytoplasmic function sigma factors (ECF σs), a major family of bacterial regulators, and determining their regulons. We generated an aligned collection of ECF σs and their promoters by leveraging the auto-regulatory nature of ECF σs as a means of promoter discovery and analyzed it to identify and characterize the conserved amino acid – nucleotide interactions that determine promoter specificity. This enabled de novo prediction of ECF σ specificity, which we combined with a statistically rigorous phylogenetic foot-printing pipeline based on precomputed orthologs to predict the direct targets of ∼67% of ECF σs. This global survey indicated that ECF σs play varied roles: some are global regulators controlling many genes throughout the genome that are important under many conditions, while others are local regulators, controlling few closely linked genes in response to specific stimuli. This analysis reveals important organizing principles of bacterial gene regulation and presents a conceptual and computational framework for deciphering gene regulatory networks.

CHARACTERISTICS OF miRNA INTERACTION WITH mRNA GENES OF T. AESTIVUM C2H2, ERF, GRAS TRANSCRIPTION FACTORS FAMILIES

The molecular mechanisms for increasing plant productivity remain poorly understood. Genes of C2H2, GRAS, ERF transcription factors (TFs) families play a key role in the physiological processes of plants, including wheat. In recent years, the important role of miRNAs in the regulation of the expression of many genes involved in the formation of productivity has been established. Wheat miRNA (mRNA-inhibiting RNA) target genes are involved in the regulation of the development of flowers, seeds, root, shoots, and responses to abiotic and biotic stresses. The miRNAs binding sites in mRNAs of C2H2, ERF, GRAS TFs families were performed using the MirTarget program, which calculates the free energy of miRNA binding with mRNA, the schemes and positions of nucleotide interactions with binding sites. Wheat genes were used as the object of the study, since wheat is one of the main grain crops in Kazakhstan and in many other countries. The presence of miRNA binding sites with high nucleotide complementarity in mRNA of C2H2, ERF, GRAS TF genes of wheat was shown. All binding sites of these miRNAs were located in the CDS of mRNA target genes. Of the 125 miRNAs of T. aestivum, miR319-3p efficiently bound with mRNA of C2H2 family genes with the value of ΔG/ΔGm equal 91 %. miR7757-5p interacted with mRNA of ERF and GRAS family genes with the value of ΔG/ΔGm equal to 92 % and 90 % respectively. miR9778-5p bound with mRNA of C2H2, ERF, GRAS family genes to varying degrees. Each of the miR408-3p, miR9780-3p, and miR9778-5p had four target genes with the value of ΔG/ΔGm equal to 87 % and 89 %. These data indicate the dependency of C2H2, GRAS, ERF TFs families expression on miRNA. The obtained results expand the fundamental ideas about the regulatory mechanisms of miRNA in the process of plant growth and development.

Binding affinity and conformational preferences influence kinetic stability of short oligonucleotides on carbon nanotubes

DNA-wrapped single walled carbon nanotubes (SWNTs) have found a widespread use in a variety of nanotechnology applications. Yet, the relationship between structural conformation, binding affinity and kinetic stability of these polymers on SWNTs remains poorly understood. Here, we used molecular dynamics (MD) simulations and experiments to explore this relationship for short oligonucleotides adsorbed on SWNTs. First, using classical MD simulations of oligonucleotide-(9,4)-SWNT hybrid complexes, we explored the relationship between ssDNA and ssRNA surface conformation and sequence chemistry. We screened the conformation of 36 sequences of short ssDNA and ssRNA polymers on (9,4) SWNT, where the contour lengths were selected so the polymers can, to a first approximation, wrap once around the SWNT circumference. From these screens, we identified structural motifs that we broadly classified into rings and non-rings. Then, several sequences were selected for detailed investigations. We used temperature replica exchange MD calculations to compute two-dimensional free energy landscapes characterizing the conformations of select sequences. Ring conformations seemed to be driven primarily by sequence chemistry. Specifically, strong (n,n+2) nucleotide interactions and the ability of the polymer to form compact structures, as for example, through sharp bends in the nucleotide backbone, correlated with ring-forming propensity. However, ring-formation probability was found to be uncorrelated with free energy of oligonucleotide binding to SWNTs (∆Gbind). Conformational analyses of oligonucleotides, computed free energy of binding of oligonucleotides to SWNTs, and experimentally determined kinetic stability measurements show that ∆Gbind is the primary correlate for kinetic stability. The probability of the sequence to adopt a compact, ring-like conformation is shown to play a secondary role that still contributes measurably to kinetic stability. For example, sequences that form stable compact rings (C-rich sequences) could compensate for their relatively lower ∆Gbind and exhibit kinetic stability, while sequences with strong ∆Gbind (such as (TG)3(GT)3) were found to be kinetically stable despite their low ring formation propensity. We conclude that the stability of adsorbed oligonucleotides is primarily driven by its free energy of binding and that if ring-like structural motifs form, they would contribute positively to stability.

DIRECT: RNA contact predictions by integrating structural patterns

Background It is widely believed that tertiary nucleotide-nucleotide interactions are essential in determining RNA structure and function. Currently, direct coupling analysis (DCA) infers nucleotide contacts in a sequence from its homologous sequence alignment across different species. DCA and similar approaches that use sequence information alone typically yield a low accuracy, especially when the available homologous sequences are limited. Therefore, new methods for RNA structural contact inference are desirable because even a single correctly predicted tertiary contact can potentially make the difference between a correct and incorrectly predicted structure. Here we present a new method DIRECT (Direct Information REweighted by Contact Templates) that incorporates a Restricted Boltzmann Machine (RBM) to augment the information on sequence co-variations with structural features in contact inference. Results Benchmark tests demonstrate that DIRECT achieves better overall performance than DCA approaches. Compared to mfDCA and plmDCA, DIRECT produces a substantial increase of 41 and 18%, respectively, in accuracy on average for contact prediction. DIRECT improves predictions for long-range contacts and captures more tertiary structural features. Conclusions We developed a hybrid approach that incorporates a Restricted Boltzmann Machine (RBM) to augment the information on sequence co-variations with structural templates in contact inference. Our results demonstrate that DIRECT is able to improve the RNA contact prediction.