Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.

Serine hydrolases have diverse intracellular substrates, biological functions, and structural plasticity, and are thus important for biocatalyst design. Amongst serine hydrolases, the recently described ybfF enzyme family are promising novel biocatalysts with an unusual bifurcated substrate-binding cleft and the ability to recognize commercially relevant substrates. We characterized in detail the substrate selectivity of a novel ybfF enzyme from Vibrio cholerae (Vc-ybfF) by using a 21-member library of fluorogenic ester substrates. We assigned the roles of the two substrate-binding clefts in controlling the substrate selectivity and folded stability of Vc-ybfF by comprehensive substitution analysis. The overall substrate preference of Vc-ybfF was for short polar chains, but it retained significant activity with a range of cyclic and extended esters. This broad substrate specificity combined with the substitutional analysis demonstrates that the larger binding cleft controls the substrate specificity of Vc-ybfF. Key selectivity residues (Tyr116, Arg120, Tyr209) are also located at the larger binding pocket and control the substrate specificity profile. In the structure of ybfF the narrower binding cleft contains water molecules prepositioned for hydrolysis, but based on substitution this cleft showed only minimal contribution to catalysis. Instead, the residues surrounding the narrow binding cleft and at the entrance to the binding pocket contributed significantly to the folded stability of Vc-ybfF. The relative contributions of each cleft of the binding pocket to the catalytic activity and folded stability of Vc-ybfF provide a valuable map for designing future biocatalysts based on the ybfF scaffold.

We present a database of repetitive DNA elements, called Dfam (http://dfam.janelia.org). Many genomes contain a large fraction of repetitive DNA, much of which is made up of remnants of transposable elements (TEs). Accurate annotation of TEs enables research into their biology and can shed light on the evolutionary processes that shape genomes. Identification and masking of TEs can also greatly simplify many downstream genome annotation and sequence analysis tasks. The commonly used TE annotation tools RepeatMasker and Censor depend on sequence homology search tools such as cross_match and BLAST variants, as well as Repbase, a collection of known TE families each represented by a single consensus sequence. Dfam contains entries corresponding to all Repbase TE entries for which instances have been found in the human genome. Each Dfam entry is represented by a profile hidden Markov model, built from alignments generated using RepeatMasker and Repbase. When used in conjunction with the hidden Markov model search tool nhmmer, Dfam produces a 2.9% increase in coverage over consensus sequence search methods on a large human benchmark, while maintaining low false discovery rates, and coverage of the full human genome is 54.5%. The website provides a collection of tools and data views to support improved TE curation and annotation efforts. Dfam is also available for download in flat file format or in the form of MySQL table dumps.

Improved electron detectors and image-processing techniques will allow the structures of macromolecules to be determined from tens of thousands of single-particle cryo-EM images, rather than the hundreds of thousands needed previously.

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus. DOI:http://dx.doi.org/10.7554/eLife.00750.001.

Mouse visual cortex is subdivided into multiple distinct, hierarchically organized areas that are interconnected through feedforward (FF) and feedback (FB) pathways. The principal synaptic targets of FF and FB axons that reciprocally interconnect primary visual cortex (V1) with the higher lateromedial extrastriate area (LM) are pyramidal cells (Pyr) and parvalbumin (PV)-expressing GABAergic interneurons. Recordings in slices of mouse visual cortex have shown that layer 2/3 Pyr cells receive excitatory monosynaptic FF and FB inputs, which are opposed by disynaptic inhibition. Most notably, inhibition is stronger in the FF than FB pathway, suggesting pathway-specific organization of feedforward inhibition (FFI). To explore the hypothesis that this difference is due to diverse pathway-specific strengths of the inputs to PV neurons we have performed subcellular Channelrhodopsin-2-assisted circuit mapping in slices of mouse visual cortex. Whole-cell patch-clamp recordings were obtained from retrobead-labeled FFV1→LM- and FBLM→V1-projecting Pyr cells, as well as from tdTomato-expressing PV neurons. The results show that the FFV1→LM pathway provides on average 3.7-fold stronger depolarizing input to layer 2/3 inhibitory PV neurons than to neighboring excitatory Pyr cells. In the FBLM→V1 pathway, depolarizing inputs to layer 2/3 PV neurons and Pyr cells were balanced. Balanced inputs were also found in the FFV1→LM pathway to layer 5 PV neurons and Pyr cells, whereas FBLM→V1 inputs to layer 5 were biased toward Pyr cells. The findings indicate that FFI in FFV1→LM and FBLM→V1 circuits are organized in a pathway- and lamina-specific fashion.

The Drosophila central brain develops from a fixed number of neuroblasts. Each neuroblast makes a clone of neurons that exhibit common trajectories. Here we identified 15 distinct clones that carry larval-born neurons innervating the Drosophila central complex (CX), which consists of four midline structures including the protocerebral bridge (PB), fan-shaped body (FB), ellipsoid body (EB), and noduli (NO). Clonal analysis revealed that the small-field CX neurons, which establish intricate projections across different CX substructures, exist in four isomorphic groups that respectively derive from four complex posterior asense-negative lineages. In terms of the region-characteristic large-field CX neurons, we found that two lineages make PB neurons, 10 lineages produce FB neurons, three lineages generate EB neurons, and two lineages yield NO neurons. The diverse FB developmental origins reflect the discrete input pathways for different FB subcompartments. Clonal analysis enlightens both development and anatomy of the insect locomotor control center.

The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARγ and promoters as a component of the core transcriptional machinery.DOI:http://dx.doi.org/10.7554/eLife.00170.001.

Clusters of time series data may change location and memberships over time; in gene expression data, this occurs as groups of genes or samples respond differently to stimuli or experimental conditions at different times. In order to uncover this underlying temporal structure, we consider dynamic clusters with time-dependent parameters which split and merge over time, enabling cluster memberships to change. These interesting time-dependent structures are useful in understanding the development of organisms or complex organs, and could not be identified using traditional clustering methods. In cell cycle data, these time-dependent structure may provide links between genes and stages of the cell cycle, whilst in developmental data sets they may highlight key developmental transitions.