Molecular mechanisms of transcription initiation—structure, function, and evolution of TFE/TFIIE-like factors and open complex formation

Transcription initiation requires that the promoter DNA is melted and the template strand is loaded into the active site of the RNA polymerase (RNAP), forming the open complex (OC). The archaeal initiation factor TFE and its eukaryotic counterpart TFIIE facilitate this process. Recent structural and biophysical studies have revealed the position of TFE/TFIIE within the pre-initiation complex (PIC) and illuminated its role in OC formation. TFE operates via allosteric and direct mechanisms. Firstly, it interacts with the RNAP and induces the opening of the flexible RNAP clamp domain, concomitant with DNA melting and template loading. Secondly, TFE binds physically to single-stranded DNA in the transcription bubble of the OC and increases its stability. The identification of the β-subunit of archaeal TFE enabled us to reconstruct the evolutionary history of TFE/TFIIE-like factors, which is characterised by winged helix (WH) domain expansion in eukaryotes and loss of metal centres including iron-sulfur clusters and Zinc ribbons. OC formation is an important target for the regulation of transcription in all domains of life. We propose that TFE and the bacterial general transcription factor CarD, although structurally and evolutionary unrelated, show interesting parallels in their mechanism to enhance OC formation. We argue that OC formation is used as a way to regulate transcription in all domains of life, and these regulatory mechanisms coevolved with the basal transcription machinery

TFE and Spt4/5 open and close the RNA polymerase clamp during the transcription cycle

Transcription is an intrinsically dynamic process and requires the coordinated interplay of RNA polymerases (RNAPs) with nucleic acids and transcription factors. Classical structural biology techniques have revealed detailed snapshots of a subset of conformational states of the RNAP as they exist in crystals. A detailed view of the conformational space sampled by the RNAP and the molecular mechanisms of the basal transcription factors E (TFE) and Spt4/5 through conformational constraints has remained elusive. We monitored the conformational changes of the flexible clamp of the RNAP by combining a fluorescently labeled recombinant 12-subunit RNAP system with single-molecule FRET measurements. We measured and compared the distances across the DNA binding channel of the archaeal RNAP. Our results show that the transition of the closed to the open initiation complex, which occurs concomitant with DNA melting, is coordinated with an opening of the RNAP clamp that is stimulated by TFE. We show that the clamp in elongation complexes is modulated by the nontemplate strand and by the processivity factor Spt4/5, both of which stimulate transcription processivity. Taken together, our results reveal an intricate network of interactions within transcription complexes between RNAP, transcription factors, and nucleic acids that allosterically modulate the RNAP during the transcription cycle

Impact of marine sand extraction on benthic communities west off Sylt (SE North Sea)

Ongoing erosion at coasts, beaches and dunes accompanied by a climate change-induced sea-level rise requires extensive protection measures. At the Island of Sylt (SE North Sea) beach nourishments were conducted for almost 50 years to protect the exposed western coast against erosion. Since 1984, the materials for the sand replenishments were dredged from an offshore excavation site approx. 7 km west off Sylt in the German Bight. In this study, we investigate the long-term effects of sand extraction on the local geomorphology, the associated benthic habitats and fauna. Hydroacoustic surveys and grab sampling revealed that after more than 35 years changes in bathymetry (with dredging pits of down to ~15 m below sea floor) and also changes in habitat characteristics are still present. Additionally, the sediment and benthic faunal compositions have changed. A comparison between dredged areas and undisturbed seafloor revealed significant differences in mud content (increasing), the number of individuals and species of macrozoobenthic organisms (decreasing). This indicates that the benthic communities in the dredging areas are in a persistent successional stage. Mud-loving species (e. g. Notomastus latericeus and Kurtiella bidentate) profit from the changed habitats, however sand-preferring organisms (e.g. Pisione remota and Aonides paucibranchiata) largely disappeared. Because of the slow backfill rates, we conclude that a complete backfill of the deep dredging pits is likely to take centuries. The same is expected to apply for the regeneration of the benthic communities. However, since rather coarse-to-medium sand was removed from this area and re-accumulation of this Pleistocene material is not possible because of weak transport rates, a re-establishment of benthic communities that prefer coarser sand seems to be unlikely. Since benthic communities are strongly linked to the habitat characteristics, habitat mapping using hydroacoustic techniques is an efficient and cost-effective measure to monitor the state of regeneration in this study site

Ranking earthquake forecasts using proper scoring rules: Binary events in a low probability environment

Operational earthquake forecasting for risk management and communication during seismic sequences depends on our ability to select an optimal forecasting model. To do this, we need to compare the performance of competing models with each other in prospective forecasting mode, and to rank their performance using a fair, reproducible and reliable method. The Collaboratory for the Study of Earthquake Predictability (CSEP) conducts such prospective earthquake forecasting experiments around the globe. One metric that has been proposed to rank competing models is the Parimutuel Gambling score, which has the advantage of allowing alarm-based (categorical) forecasts to be compared with probabilistic ones. Here we examine the suitability of this score for ranking competing earthquake forecasts. First, we prove analytically that this score is in general improper, meaning that, on average, it does not prefer the model that generated the data. Even in the special case where it is proper, we show it can still be used in an improper way. Then, we compare its performance with two commonly-used proper scores (the Brier and logarithmic scores), taking into account the uncertainty around the observed average score. We estimate the confidence intervals for the expected score difference which allows us to define if and when a model can be preferred. Our findings suggest the Parimutuel Gambling score should not be used to distinguishing between multiple competing forecasts. They also enable a more rigorous approach to distinguish between the predictive skills of candidate forecasts in addition to their rankings.Comment: 29 pages, 14 figures. Work presented at vEGU21 as vPico presentatio

Crystal structure and RNA binding of the Rpb4/Rpb7 subunits of human RNA polymerase II

The Rpb4 and Rpb7 subunits of eukaryotic RNA polymerase II (RNAP(II)) form a heterodimer that protrudes from the 10-subunit core of the enzyme. We have obtained crystals of the human Rpb4/Rpb7 heterodimer and determined the structure to 2.7 Å resolution. The presence of putative RNA-binding domains on the Rpb7 subunit and the position of the heterodimer close to the RNA exit groove in the 12 subunit yeast polymerase complex strongly suggests a role for the heterodimer in binding and stabilizing the nascent RNA transcript. We have complemented the structural analysis with biochemical studies directed at dissecting the RNA-binding properties of the human Rpb4/Rpb7 complex and that of the homologous E/F complex from Methanocaldococcus jannaschii. A number of conserved, solvent-exposed residues in both the human Rpb7 subunit and the archaeal E subunit have been modified by site-directed mutagenesis and the mutants tested for RNA binding by performing electrophoretic mobility shift assays. These studies have identified an elongated surface region on the corresponding face of both subunit E and Rpb7 that is involved in RNA binding. The area spans the nucleic acid binding face of the OB fold, including the B4–B5 loop, but also extends towards the N-terminal domain

Persistent effects of sand extraction on habitats and associated benthic communities in the German Bight

Sea-level rise demands for protection measures of endangered coastlines crucial for the local population. At the island of Sylt in the SE North Sea, shoreline erosion is compensated by replenishment with sand dredged from an offshore extraction site. We studied the long-term effects of sand extraction on bathymetry, geomorphology, habitats and benthic fauna. Sand extraction created dredging holes about 1 km in diameter and up to 20 m below the ambient seafloor level. Directly after dredging the superficial sediment layer, inside the pits was dominated by coarse sand and stones. Hydroacoustic surveys revealed only minor changes of bathymetry > 35 years after sand extraction. Obviously, backfill of the dredging pits was very slow, at a rate of a few millimeters per year, presumably resulting from low ambient sediment availability and relatively calm hydrodynamic conditions despite high wave energy during storms. Thus, a complete backfill of the deep extraction sites is likely to take centuries in this area. Hydroacoustic surveys and ground truthing showed that the backfilled material is mainly very fine sand and mud, turning the previously coarse sand surface into a muddy habitat. Accordingly, grab samples revealed significant differences in macrozoobenthos community composition, abundance and species density between recently dredged areas ( 10 years ago) and undisturbed sites (control sites). Overall, dredging turned the original association of sand-dwelling species into a muddy sediment association. Since re-establishment of disturbed benthic communities depends on previous re-establishment of habitat characteristics, the low sedimentation rates indicate that a return to a pre-dredging habitat type with its former benthic community and habitat characteristics is unlikely. Since coarse sand is virtually immobile in this area, a regeneration towards pre-dredging conditions is also unlikely without human interference (e.g., mitigation measures like depositing coarse material on the seafloor to restore the sessile epifauna)

Proteome Analysis of Swine Macrophages after Infection with Two Genotype II African Swine Fever Isolates of Different Pathogenicity

Since the introduction of a highly pathogenic genotype II isolate of the African swine fever virus (ASFV) into Georgia in 2007, African swine fever (ASF) has gone panzootic. Outbreaks have been reported in Europe, Asia and, more recently, Latin America. Thus, ASFV has become a major threat to the pig industry worldwide, as broadly applicable vaccines are not available. While the majority of ASFV strains show high virulence in domestic pigs and wild boar, variations within the ASFV genome have resulted in the emergence of attenuated strains with low or moderate virulence. However, the molecular basis of the differences in virulence has not yet been discovered. To reveal virulence-associated protein expression patterns, we analysed the proteomes of the natural target cells of ASFV, primary porcine macrophages, after infection with two genotype II ASFV strains displaying high (Armenia 2008) and moderate (Estonia 2014) virulence using quantitative mass spectrometry. Very similar expression patterns were observed for the viral genes, and any differences were limited to the deletions within the Estonia 2014 genome. In addition to the canonical ASFV proteins, twelve novel protein products from recently described transcripts were confirmed in both isolates. Pathway analyses showed that both isolates evoked a similar host proteome response, despite their difference in virulence. However, subtle differences in the manipulation of the proteins involved in the proinflammatory response mediated by the MAPK14/p38 signalling cascade were observe

In Situ Experiments To Reveal the Role of Surface Feature Sidewalls in the Cassie–Wenzel Transition

Waterproof and self-cleaning surfaces continue to attract much attention as they can be instrumental in various different technologies. Such surfaces are typically rough, allowing liquids to contact only the outermost tops of their asperities, with air being entrapped underneath. The formed solid–liquid–air interface is metastable and, hence, can be forced into a completely wetted solid surface. A detailed understanding of the wetting barrier and the dynamics of this transition is critically important for the practical use of the related surfaces. Toward this aim, wetting transitions were studied in situ at a set of patterned perfluoropolyether dimethacrylate (PFPEdma) polymer surfaces exhibiting surface features with different types of sidewall profiles. PFPEdma is intrinsically hydrophobic and exhibits a refractive index very similar to water. Upon immersion of the patterned surfaces into water, incident light was differently scattered at the solid–liquid–air and solid–liquid interface, which allows for distinguishing between both wetting states by dark-field microscopy. The wetting transition observed with this methodology was found to be determined by the sidewall profiles of the patterned structures. Partial recovery of the wetting was demonstrated to be induced by abrupt and continuous pressure reductions. A theoretical model based on Laplace’s law was developed and applied, allowing for the analytical calculation of the transition barrier and the potential to revert the wetting upon pressure reduction