Mycobacterium tuberculosis CarD, an essential global transcriptional regulator forms amyloid-like fibrils

CarD is an essential global transcription regulator from Mycobacterium tuberculosis (Mtb) that binds RNA polymerase and activates transcription by stabilizing the transcription initiation complex. Available crystal structures have captured two distinct, monomeric and domain-swapped homodimeric, oligomeric states of CarD. However, the actual oligomeric state of CarD in solution and its biological relevance has remained unclear. Here, we confirm the presence of the homodimeric state of CarD in solution by using synchrotron-based small-angle X-ray scattering. Furthermore, by using biochemical and biophysical experiments, in addition to mass-spectrometry, transmission electron microscopy, and confocal imaging, we show that CarD is the first soluble cytosolic protein in Mtb which displays the tendency to form amyloid-like fibrils both in vitro as well as in vivo. We demonstrate that the deletion of the fourteen N-terminal residues involved in domain-swapping hampers amyloid formation, thus, suggesting that domain-swapping is crucial in amyloidogenesis. The discovery of the amyloidogenic property of an essential cytosolic global transcription regulator, CarD, in a pathogenic bacteria will further open up new frontiers in research.Peer reviewe

Crystallization and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77

The major capsid proteins VP16 and VP17 of bacteriophage P23-77 have been crystallized using both recombinant and purified virus and preliminary diffraction analyses have been performed

The Structure of an RNAi Polymerase Links RNA Silencing and Transcription

RNA silencing refers to a group of RNA-induced gene-silencing mechanisms that developed early in the eukaryotic lineage, probably for defence against pathogens and regulation of gene expression. In plants, protozoa, fungi, and nematodes, but apparently not insects and vertebrates, it involves a cell-encoded RNA-dependent RNA polymerase (cRdRP) that produces double-stranded RNA triggers from aberrant single-stranded RNA. We report the 2.3-Å resolution crystal structure of QDE-1, a cRdRP from Neurospora crassa, and find that it forms a relatively compact dimeric molecule, each subunit of which comprises several domains with, at its core, a catalytic apparatus and protein fold strikingly similar to the catalytic core of the DNA-dependent RNA polymerases responsible for transcription. This evolutionary link between the two enzyme types suggests that aspects of RNA silencing in some organisms may recapitulate transcription/replication pathways functioning in the ancient RNA-based world

A Deep HST Search for Escaping Lyman Continuum Flux at z~1.3: Evidence for an Evolving Ionizing Emissivity

We have obtained deep Hubble Space Telescope far-UV images of 15 starburst galaxies at z~1.3 in the GOODS fields to search for escaping Lyman continuum photons. These are the deepest far-UV images m_{AB}=28.7, 3\sigma, 1" diameter) over this large an area (4.83 arcmin^2) and provide the best escape fraction constraints for any galaxy at any redshift. We do not detect any individual galaxies, with 3\sigma limits to the Lyman Continuum (~700 \AA) flux 50--149 times fainter (in f_nu) than the rest-frame UV (1500 \AA) continuum fluxes. Correcting for the mean IGM attenuation (factor ~2), as well as an intrinsic stellar Lyman Break (~3), these limits translate to relative escape fraction limits of f_{esc,rel}<[0.03,0.21]. The stacked limit is f_{esc,rel}(3\sigma)<0.02. We use a Monte Carlo simulation to properly account for the expected distribution of IGM opacities. When including constraints from previous surveys at z~1.3 we find that, at the 95% confidence level, no more than 8% of star--forming galaxies at z~1.3 can have relative escape fractions greater than 0.50. Alternatively, if the majority of galaxies have low, but non-zero, escaping Lyman Continuum, the escape fraction can not be more than 0.04. Both the stacked limits, and the limits from the Monte Carlo simulation suggest that the average ionizing emissivity (relative to non-ionizing UV emissivity) at z~1.3 is significantly lower than has been observed in Lyman Break Galaxies (LBGs) at z~3. If the ionizing emissivity of star-forming galaxies is in fact increasing with redshift, it would help to explain the high photoionization rates seen in the IGM at z>4 and reionization of the intergalactic medium at z>6. [Abridged]Comment: Submitted to ApJ (Nov. 6) Comments Welcome. 11 pages, 8 figure

Insights into the pre-initiation events of bacteriophage phi6 RNA-dependent RNA polymerase : towards the assembly of a productive binary complex

The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.The RNA-dependent RNA polymerase (RdRP) of double-stranded RNA (dsRNA) viruses performs both RNA replication and transcription. In order to initiate RNA polymerization, viral RdRPs must be able to interact with the incoming 3 terminus of the template and position it, so that a productive binary complex is formed. Structural studies have revealed that RdRPs of dsRNA viruses that lack helicases have electrostatically charged areas on the polymerase surface, which might facilitate such interactions. In this study, structure-based mutagenesis, enzymatic assays and molecular mapping of bacteriophage 6 RdRP and its RNA were used to elucidate the roles of the negatively charged plough area on the polymerase surface, of the rim of the template tunnel and of the template specificity pocket that is key in the formation of the productive RNA-polymerase binary complex. The positively charged rim of the template tunnel has a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. Hence, we show that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the 6 RdRP can be greatly enhanced.Peer reviewe

Accelerated volume loss in glacier ablation zones of NE Greenland, Little Ice Age to present

Mountain glaciers at the periphery of the Greenland ice sheet are a crucial freshwater and sediment source to the North Atlantic and strongly impact Arctic terrestrial, fjord, and coastal biogeochemical cycles. In this study we mapped the extent of 1,848 mountain glaciers in NE Greenland at the Little Ice Age. We determined area and volume changes for the time periods Little Ice Age to 1980s and 1980s to 2014 and equilibrium line altitudes. There was at least 172.76 ± 34.55‐km3 volume lost between 1910 and 1980s, that is, a rate of 2.61 ± 0.52 km3/year. Between 1980s and 2014 the volume lost was 90.55 ± 18.11 km3, that is, a rate of 3.22 ± 0.64 km3/year, implying an increase of ~23% in the rate of ice volume loss. Overall, at least ~7% of mass loss from Greenland mountain glaciers and ice caps has come from the NE sector

Accelerated volume loss in glacier ablation zones of NE Greenland, Little Ice Age to present

Mountain glaciers at the periphery of the Greenland ice sheet are a crucial freshwater and sediment source to the North Atlantic and strongly impact Arctic terrestrial, fjord, and coastal biogeochemical cycles. In this study we mapped the extent of 1,848 mountain glaciers in NE Greenland at the Little Ice Age. We determined area and volume changes for the time periods Little Ice Age to 1980s and 1980s to 2014 and equilibrium line altitudes. There was at least 172.76 ± 34.55‐km3 volume lost between 1910 and 1980s, that is, a rate of 2.61 ± 0.52 km3/year. Between 1980s and 2014 the volume lost was 90.55 ± 18.11 km3, that is, a rate of 3.22 ± 0.64 km3/year, implying an increase of ~23% in the rate of ice volume loss. Overall, at least ~7% of mass loss from Greenland mountain glaciers and ice caps has come from the NE sector

Towards in cellulo virus crystallography

Viruses are a significant threat to both human health and the economy, and there is an urgent need for novel anti-viral drugs and vaccines. High-resolution viral structures inform our understanding of the virosphere, and inspire novel therapies. Here we present a method of obtaining such structural information that avoids potentially disruptive handling, by collecting diffraction data from intact infected cells. We identify a suitable combination of cell type and virus to accumulate particles in the cells, establish a suitable time point where most cells contain virus condensates and use electron microscopy to demonstrate that these are ordered crystalline arrays of empty capsids. We then use an X-ray free electron laser to provide extremely bright illumination of sub-micron intracellular condensates of bacteriophage phiX174 inside living Escherichia coli at room temperature. We have been able to collect low resolution diffraction data. Despite the limited resolution and completeness of these initial data, due to a far from optimal experimental setup, we have used novel methodology to determine a putative space group, unit cell dimensions, particle packing and likely maturation state of the particles.Peer reviewe

Crystal structure and carbohydrate analysis of Nipah virus attachment glycoprotein:a template for antiviral and vaccine design

Two members of the paramyxovirus family, Nipah virus (NiV) and Hendra virus (HeV), are recent additions to a growing number of agents of emergent diseases which use bats as a natural host. Identification of ephrin-B2 and ephrin-B3 as cellular receptors for these viruses has enabled the development of immunotherapeutic reagents which prevent virus attachment and subsequent fusion. Here we present the structural analysis of the protein and carbohydrate components of the unbound viral attachment glycoprotein of NiV glycoprotein (NiV-G) at a 2.2-A resolution. Comparison with its ephrin-B2-bound form reveals that conformational changes within the envelope glycoprotein are required to achieve viral attachment. Structural differences are particularly pronounced in the 579-590 loop, a major component of the ephrin binding surface. In addition, the 236-245 loop is rather disordered in the unbound structure. We extend our structural characterization of NiV-G with mass spectrometric analysis of the carbohydrate moieties. We demonstrate that NiV-G is largely devoid of the oligomannose-type glycans that in viruses such as human immunodeficiency virus type 1 and Ebola virus influence viral tropism and the host immune response. Nevertheless, we find putative ligands for the endothelial cell lectin, LSECtin. Finally, by mapping structural conservation and glycosylation site positions from other members of the paramyxovirus family, we suggest the molecular surface involved in oligomerization. These results suggest possible pathways of virus-host interaction and strategies for the optimization of recombinant vaccines