Search

Archaeal transcription is carried out by a multi-subunit RNA polymerase (RNAP) that is highly homologous in structure and function to eukaryotic RNAP II. Among the set of basal transcription factors, only Spt5 is found in all domains of life, but Spt5 has been shaped during evolution, which is also reflected in the heterodimerization of Spt5 with Spt4 in Archaea and Eukaryotes. To unravel the mechanistic basis of Spt4/5 function in Archaea, we performed structure-function analyses using the archaeal transcriptional machinery of Pyrococcus furiosus (Pfu). We report single-particle cryo-electron microscopy reconstructions of apo RNAP and the archaeal elongation complex (EC) in the absence and presence of Spt4/5. Surprisingly, Pfu Spt4/5 also binds the RNAP in the absence of nucleic acids in a distinct super-contracted conformation. We show that the RNAP clamp/stalk module exhibits conformational flexibility in the apo state of RNAP and that the enzyme contracts upon EC formation or Spt4/5 engagement. We furthermore identified a contact of the Spt5-NGN domain with the DNA duplex that stabilizes the upstream boundary of the transcription bubble and impacts Spt4/5 activity in vitro. This study, therefore, provides the structural basis for Spt4/5 function in archaeal transcription and reveals a potential role beyond the well-described support of elongation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: People with intellectual disabilities may experience frailty earlier than the general population. This scoping review aimed to investigate how frailty is defined, assessed, and managed in adults with an intellectual disability; factors associated with frailty; and the potential impact of COVID-19 on frailty identification and management., Method: Databases were searched from January 2016 to July 2023 for studies that investigated frailty in individuals with intellectual disabilities., Results: Twenty studies met the inclusion criteria. Frailty prevalence varied between 9% and 84%. Greater severity of intellectual disability, presence of Down syndrome, older age, polypharmacy, and group home living were associated with frailty. Multiagency working, trusted relationships and provision of evidence-based information may all be beneficial in frailty management., Conclusion: Frailty is common for people with intellectual disabilities and is best identified with measures specifically designed for this population. Future research should evaluate interventions to manage frailty and improve lives., (© 2024 The Authors. Journal of Applied Research in Intellectual Disabilities published by John Wiley & Sons Ltd.)

Over the last years, single-molecule force spectroscopy provided insights into the intricate connection between mechanical stimuli and biochemical signaling. The underlying molecular mechanisms were uncovered and explored using techniques such as atomic force microscopy and force spectroscopy using optical or magnetic tweezers. These experimental approaches are limited by thermal noise resulting from a physical connection of the studied biological system to the macroscopic world. To overcome this limitation, we recently introduced the DNA origami force clamp (FC) which is a freely diffusing nanodevice that generates piconewton forces on a DNA sequence of interest. Binding of a protein to the DNA under tension can be detected employing fluorescence resonance energy transfer (FRET) as a sensitive readout.This protocol introduces the reader to the working principles of the FC and provides instructions to design and generate a DNA origami FC customized for a protein of interest. Molecular cloning techniques are employed to modify, produce, and purify a custom DNA scaffold. A fluorescently labeled DNA suitable to detect protein binding via FRET is generated via enzymatic ligation of commercial DNA oligonucleotides. After thermal annealing of all components, the DNA origami FC is purified using agarose gel electrophoresis. The final section covers the interrogation of the FC using confocal single-molecule FRET measurements and subsequent data analysis to quantify the binding of a DNA-binding protein to its cognate recognition site under a range of forces. Using this approach, force-dependent DNA-protein interactions can be studied on the single-molecule level on thousands of molecules in a parallelized fashion., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: Extreme environments provide unique challenges for life, and the study of extremophiles can shed light on the mechanisms of adaptation to such conditions. Pyrococcus furiosus , a hyperthermophilic archaeon, is a model organism for studying thermal stress response mechanisms. In this study, we used an integrated analysis of RNA-sequencing and mass spectrometry data to investigate the transcriptomic and proteomic responses of P. furiosus to heat and cold shock stress and recovery. Our results reveal the rapid and dynamic changes in gene and protein expression patterns associated with these stress responses, as well as the coordinated regulation of different gene sets in response to different stressors. These findings provide valuable insights into the molecular adaptations that facilitate life in extreme environments and advance our understanding of stress response mechanisms in hyperthermophilic archaea., Competing Interests: The authors declare no competing interests. D.G. and G.S. are co-founders of Microbify GmbH. However, this study was not funded by Microbify GmbH.

Objectives: This review explored the literature on the use of social media in recruiting young people, aged 13-18 years, to mental health research. It aimed to identify barriers and facilitators to recruitment and strategies to improve participation in future research., Design: Scoping review., Data Sources: Articles published between January 2011 and February 2023 were searched for on PubMed, Scopus, Medline (via EBSCOhost) and Cochrane Library databases., Eligibility Criteria: Studies that outlined social media as a recruitment method and recruited participants aged 13-18 years., Data Extraction and Synthesis: Data was extracted by two reviewers independently and cross-checked by a third reviewer. Data on study design, aims, participants, recruitment methods and findings related specifically to social media as a recruitment tool were collected., Results: 24 journal articles met the inclusion criteria. Studies were predominantly surveys (n=13) conducted in the USA (n=16) recruiting via Facebook (n=16) and/or Instagram (n=14). Only nine of the included articles provided a summary of success and reviewed the efficacy of social media recruitment for young people in mental health research. Type of advertisement, the language used, time of day and the use of keywords were all found to be factors that may influence the success of recruitment through social media; however, as these are based on findings from a small number of studies, such potential influences require further investigation., Conclusion: Social media recruitment can be a successful method for recruiting young people to mental health research. Further research is needed into recruiting socioeconomically marginalised groups using this method, as well as the effectiveness of new social media platforms., Registration: Open Science Framework Registry (https://osf.io/mak75/)., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

The regulation of archaellation, the formation of archaeal-specific cell appendages called archaella, is crucial for the motility, adhesion, and survival of archaeal organisms. Although the heavily archaellated and highly motile Pyrococcus furiosus is a key model organism for understanding the production and function of archaella in Euryarchaea, the transcriptional regulation of archaellum assembly is so far unknown. Here we show that the transcription factor EarA is the master regulator of the archaellum ( arl ) operon transcription, which is further modulated by intergenic transcription termination signals. EarA deletion or overexpression strains demonstrate that EarA is essential for archaellation in P. furiosus and governs the degree of archaellation. Providing a single-molecule update on the transcriptional landscape of the arl operon in P. furiosus , we identify sequence motifs for EarA binding upstream of the arl operon and intergenic terminator sequences as critical elements for fine-tuning the expression of the multicistronic arl cluster. Furthermore, transcriptome re-analysis across different Thermococcales species demonstrated a heterogeneous production of major archaellins, suggesting a more diverse composition of archaella than previously recognized. Overall, our study provides novel insights into the transcriptional regulation of archaellation and highlights the essential role of EarA in Pyrococcus furiosus . These findings advance our understanding of the mechanisms governing archaellation and have implications for the functional diversity of archaella., Competing Interests: DG is co-founder of Microbify GmbH. However, there are no commercial interests by the company, or any financial support granted by Microbify GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Stöckl, Nißl, Reichelt, Rachel, Grohmann and Grünberger.)

Importance: Heat shock response is the ability to respond adequately to sudden temperature increases that could be harmful for cellular survival and fitness. It is crucial for microorganisms living in volcanic hot springs that are characterized by high temperatures and large temperature fluctuations. In this study, we investigated how S. acidocaldarius , which grows optimally at 75°C, responds to heat shock by altering its gene expression and protein production processes. We shed light on which cellular processes are affected by heat shock and propose a hypothesis on underlying regulatory mechanisms. This work is not only relevant for the organism's lifestyle, but also with regard to its evolutionary status. Indeed, S. acidocaldarius belongs to the archaea, an ancient group of microbes that is more closely related to eukaryotes than to bacteria. Our study thus also contributes to a better understanding of the early evolution of heat shock response., Competing Interests: The authors declare no conflict of interest.

Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level., (© 2023 the author(s), published by De Gruyter, Berlin/Boston.)

Objective: This systematic review and meta-analysis assessed the efficacy of exercise in reducing OCD symptoms., Methods: We searched PubMed, Cochrane Central Register of Controlled Trials, MEDLINE, Scopus and grey literature until March 2022. The study was preregistered at Prospero (CRD42021283931). We included randomised controlled and pre-post trials assessing physical activity as an intervention for OCD. Risk of bias was assessed using the Cochrane ROBINS-I tool and the RoB2 tool., Results: The analysis included 6 trials ( N  = 92); 2 were RCTS and 4 were pre-post design studies. A random-effects meta-analysis of pre-post data identified a large reduction of OCD symptoms following exercise ( g  = 1.33 [95%CI 1.06-1.61]; k  = 6). Exercise was also associated with significant pre-post reductions in anxiety ( g  = 0.71 [95%CI 0.37-1.05; k  = 4) and depression ( g  = 0.57 [95%CI 0.26-0.89]; k  = 2). Risk of bias was moderate-high in uncontrolled trials on the ROBINS-I and RCTs showed 'some concerns' on the RoB2., Conclusion: Exercise was associated with a large pre-post reduction of OCD symptoms; however, few trials were of robust quality and all were at risk of bias. Further well-powered and better quality RCTs are required to assess the role of exercise as an intervention for OCD.KEY POINTSStudies exploring exercise as an adjunct therapy for OCD have small participant numbers, therefore a systematic review and meta-analysis is needed to estimate potential efficacy.Pre-post analysis shows that exercise was associated with a large reduction of OCD symptomsThe current systematic review and meta-analysis points to the potential for exercise to be beneficial for the treatment for OCD symptoms. However, more well-powered and better controlled RCTs are required to fully assess the benefit of exercise for the treatment of OCD symptoms.

Surface-layer (S-layer) proteins form the outermost envelope in many bacteria and most archaea and arrange in two-dimensional quasicrystalline structures via self-assembly. We investigated S-layer proteins extracted from the archaeon Pyrobaculum aerophilium with a qPlus sensor-based atomic force microscope (AFM) in both liquid and ambient conditions and compared it to transmission electron microscopy (TEM) images under vacuum conditions. For AFM scanning, a next-generation liquid cell and a new protocol for creating long and sharp sapphire tips was introduced. Initial AFM images showed only layers of residual detergent molecules (sodium dodecyl sulfate, SDS), which are used to isolate the S-layer proteins from the cells. SDS was not visible in the TEM images, requiring more thorough sample preparation for AFM measurements. These improvements allowed us to resolve the crystallike structure of the S-layer samples with frequency-modulation AFM in both air and liquid.

Ribosomal RNA (rRNA) maturation in archaea is a complex multistep process that requires well-defined endo- and exoribonuclease activities to generate fully mature linear rRNAs. However, technical challenges prevented detailed mapping of rRNA processing steps and a systematic analysis of rRNA maturation pathways across the tree of life. In this study, we used long-read (PCR)-cDNA and direct RNA nanopore-based sequencing to study rRNA maturation in three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius Compared to standard short-read protocols, nanopore sequencing facilitates simultaneous readout of 5'- and 3'-positions, which is required for the classification of rRNA processing intermediates. More specifically, we (i) accurately detect and describe rRNA maturation stages by analysis of terminal read positions of cDNA reads and thereupon (ii) explore the stage-dependent installation of the KsgA-mediated dimethylations in H. volcanii using base-calling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of nanopore sequencing, we could detect hitherto unknown intermediates with high confidence, revealing details about the maturation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features of rRNA processing in euryarchaeal and crenarchaeal representatives, thereby significantly expanding our understanding of rRNA maturation pathways in archaea., (© 2023 Grünberger et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

A novel interdomain consortium composed of a methanogenic Archaeon and a sulfate-reducing bacterium was isolated from a microbial biofilm in an oil well in Cahuita National Park, Costa Rica. Both organisms can be grown in pure culture or as stable co-culture. The methanogenic cells were non-motile rods producing CH 4 exclusively from H 2 /CO 2 . Cells of the sulfate-reducing partner were motile rods forming cell aggregates. They utilized hydrogen, lactate, formate, and pyruvate as electron donors. Electron acceptors were sulfate, thiosulfate, and sulfite. 16S rRNA sequencing revealed 99% gene sequence similarity of strain CaP3V-M-L2A T to Methanobacterium subterraneum and 98.5% of strain CaP3V-S-L1A T to Desulfomicrobium baculatum. Both strains grew from 20 to 42 °C, pH 5.0-7.5, and 0-4% NaCl. Based on our data, type strains CaP3V-M-L2A T (= DSM 113354  T  = JCM 39174  T ) and CaP3V-S-L1A T (= DSM 113299  T  = JCM 39179  T ) represent novel species which we name Methanobacterium cahuitense sp. nov. and Desulfomicrobium aggregans sp. nov., (© 2023. The Author(s).)

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology., (© 2023. The Author(s).)

Nucleases derived from the prokaryotic defense system CRISPR-Cas are frequently re-purposed for gene editing and molecular diagnostics. Hence, an in-depth understanding of the molecular mechanisms of these enzymes is of crucial importance. We focused on Cas12a from Francisella novicida (FnCas12a) and investigated the functional role of helix 1, a structural element that together with the bridge helix (BH) connects the recognition and the nuclease lobes of FnCas12a. Helix 1 is structurally connected to the lid domain that opens upon DNA target loading thereby activating the active site of FnCas12a. We probed the structural states of FnCas12a variants altered in helix 1 and/or the bridge helix using single-molecule FRET measurements and assayed the pre-crRNA processing, cis- and trans-DNA cleavage activity. We show that helix 1 and not the bridge helix is the predominant structural element that confers conformational stability of FnCas12a. Even small perturbations in helix 1 lead to a decrease in DNA cleavage activity while the structural integrity is not affected. Our data, therefore, implicate that the concerted remodeling of helix 1 and the bridge helix upon DNA binding is structurally linked to the opening of the lid and therefore involved in the allosteric activation of the active site., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A novel methanogenic strain, CaP3V-MF-L2A T , was isolated from an exploratory oil well from Cahuita National Park, Costa Rica. The cells were irregular cocci, 0.8-1.8 μm in diameter, stained Gram-negative and were motile. The strain utilized H 2 /CO 2 , formate and the primary and secondary alcohols 1-propanol and 2-propanol for methanogenesis, but not acetate, methanol, ethanol, 1-butanol or 2-butanol. Acetate was required as carbon source. The novel isolate grew at 25-40 °C, pH 6.0-7.5 and 0-2.5% (w/v) NaCl. 16S rRNA gene sequence analysis revealed that the strain is affiliated to the genus Methanofollis. It shows 98.8% sequence similarity to its closest relative Methanofollis ethanolicus. The G + C content is 60.1 mol%. Based on the data presented here type strain CaP3V-MF-L2A T (= DSM 113321 T  = JCM 39176 T ) represents a novel species, Methanofollis propanolicus sp. nov., (© 2022. The Author(s).)

Human Argonaute 2 (hAgo2) constitutes the functional core of the RNA interference pathway. Guide RNAs direct hAgo2 to target mRNAs, which ultimately leads to hAgo2-mediated mRNA degradation or translational inhibition. Here, we combine site-specifically labeled hAgo2 with time-resolved single-molecule FRET measurements to monitor conformational states and dynamics of hAgo2 and hAgo2-RNA complexes in solution that remained elusive so far. We observe dynamic anchoring and release of the guide's 3'-end from the PAZ domain during the stepwise target loading process even with a fully complementary target. We find differences in structure and dynamic behavior between partially and fully paired canonical hAgo2-guide/target complexes and the miRNA processing complex formed by hAgo2 and pre-miRNA451. Furthermore, we detect a hitherto unknown conformation of hAgo2-guide/target complexes that poises them for target-directed miRNA degradation. Taken together, our results show how the conformational flexibility of hAgo2-RNA complexes determines function and the fate of the ribonucleoprotein particle., (© 2022. The Author(s).)

Background: Research into paranormal beliefs and cognitive functioning has expanded considerably since the last review almost 30 years ago, prompting the need for a comprehensive review. The current systematic review aims to identify the reported associations between paranormal beliefs and cognitive functioning, and to assess study quality., Method: We searched four databases (Scopus, ScienceDirect, SpringerLink, and OpenGrey) from inception until May 2021. Inclusion criteria comprised papers published in English that contained original data assessing paranormal beliefs and cognitive function in healthy adult samples. Study quality and risk of bias was assessed using the Appraisal tool for Cross-Sectional Studies (AXIS) and results were synthesised through narrative review. The review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was preregistered as part of a larger registration on the Open Science Framework (https://osf.io/uzm5v)., Results: From 475 identified studies, 71 (n = 20,993) met our inclusion criteria. Studies were subsequently divided into the following six categories: perceptual and cognitive biases (k = 19, n = 3,397), reasoning (k = 17, n = 9,661), intelligence, critical thinking, and academic ability (k = 12, n = 2,657), thinking style (k = 13, n = 4,100), executive function and memory (k = 6, n = 810), and other cognitive functions (k = 4, n = 368). Study quality was rated as good-to-strong for 75% of studies and appears to be improving across time. Nonetheless, we identified areas of methodological weakness including: the lack of preregistration, discussion of limitations, a-priori justification of sample size, assessment of nonrespondents, and the failure to adjust for multiple testing. Over 60% of studies have recruited undergraduates and 30% exclusively psychology undergraduates, which raises doubt about external validity. Our narrative synthesis indicates high heterogeneity of study findings. The most consistent associations emerge for paranormal beliefs with increased intuitive thinking and confirmatory bias, and reduced conditional reasoning ability and perception of randomness., Conclusions: Although study quality is good, areas of methodological weakness exist. In addressing these methodological issues, we propose that authors engage with preregistration of data collection and analysis procedures. At a conceptual level, we argue poorer cognitive performance across seemingly disparate cognitive domains might reflect the influence of an over-arching executive dysfunction., Competing Interests: The authors have declared that no competing interests exist.

High-throughput sequencing dramatically changed our view of transcriptome architectures and allowed for ground-breaking discoveries in RNA biology. Recently, sequencing of full-length transcripts based on the single-molecule sequencing platform from Oxford Nanopore Technologies (ONT) was introduced and is widely used to sequence eukaryotic and viral RNAs. However, experimental approaches implementing this technique for prokaryotic transcriptomes remain scarce. Here, we present an experimental and bioinformatic workflow for ONT RNA-seq in the bacterial model organism Escherichia coli , which can be applied to any microorganism. Our study highlights critical steps of library preparation and computational analysis and compares the results to gold standards in the field. Furthermore, we comprehensively evaluate the applicability and advantages of different ONT-based RNA sequencing protocols, including direct RNA, direct cDNA, and PCR-cDNA. We find that (PCR)-cDNA-seq offers improved yield and accuracy compared to direct RNA sequencing. Notably, (PCR)-cDNA-seq is suitable for quantitative measurements and can be readily used for simultaneous and accurate detection of transcript 5' and 3' boundaries, analysis of transcriptional units, and transcriptional heterogeneity. In summary, based on our comprehensive study, we show nanopore RNA-seq to be a ready-to-use tool allowing rapid, cost-effective, and accurate annotation of multiple transcriptomic features. Thereby nanopore RNA-seq holds the potential to become a valuable alternative method for RNA analysis in prokaryotes., (© 2022 Grünberger et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

CRISPR-Cas9 is a ribonucleoprotein complex that sequence-specifically binds and cleaves double-stranded DNA. Wildtype Cas9 and its nickase and cleavage-incompetent mutants have been used in various biological techniques due to their versatility and programmable specificity. Cas9 has been shown to bind very stably to DNA even after cleavage of the individual DNA strands, inhibiting further turnovers and considerably slowing down in-vivo repair processes. This poses an obstacle in genome editing applications. Here, we employed single-molecule magnetic tweezers to investigate the binding stability of different Streptococcus pyogenes Cas9 variants after cleavage by challenging them with supercoiling. We find that different release mechanisms occur depending on which DNA strand is cleaved. After initial target strand cleavage, supercoils are only removed after the collapse of the R-loop. We identified several states with different stabilities of the R-loop. Most importantly, we find that the post-cleavage state of Cas9 exhibits a higher stability than the pre-cleavage state. After non-target strand cleavage, supercoils are immediately but slowly released by swiveling of the non-target strand around Cas9 bound to the target strand. Consequently, Cas9 and its non-target strand nicking mutant stay stably bound to the DNA for many hours even at elevated torsional stress., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Anxiety is common in youth on the autism spectrum and cognitive behavioural therapy (CBT) has been adapted to address associated symptoms. The aim of the current systematic review and meta-analysis was to examine the efficacy of CBT for reducing anxiety in autistic youth., Method: Searches of PubMed and Scopus databases were undertaken from January 1990 until December 2020. Studies were included if they consisted of randomised controlled trials (RCTs) using CBT to reduce anxiety in autistic youth. Separate random effects meta-analyses assessed anxiety ratings according to informant (clinician; parent; child), both at end-of-trial and at follow-up., Results: A total of 19 RCTs met our inclusion criteria (833 participants: CBT N = 487; controls N = 346). Random effects meta-analyses revealed a large effect size for clinician rated symptoms (g = 0.88, 95% CI 0.55, 1.12, k = 11), while those for both parent (g = 0.40, 95% CI 0.24, 0.56; k = 18) and child-reported anxiety (g = 0.25, 95% CI 0.06, 0.43; k = 13) were smaller, but significant. These benefits were not however maintained at follow-up. Moderator analyses showed that CBT was more efficacious for younger children (for clinician and parent ratings) and when delivered as individual therapy (for clinician ratings). Using the Cochrane Risk of Bias 2 tool, we found concerns about reporting bias across most trials., Conclusions: The efficacy of CBT for anxiety in autistic youth was supported in the immediate intervention period. However, substantial inconsistency emerged in the magnitude of benefit depending upon who was rating symptoms (clinician, parent or child). Follow-up analyses failed to reveal sustained benefits, though few studies have included this data. It will be important for future trials to address robustness of treatment gains overtime and to further explore inconsistency in efficacy by informant. We also recommend pre-registration of methods by trialists to address concerns with reporting bias., (© 2021. The Author(s).)

Objective: Mindfulness-based interventions (MBIs) are being increasingly used as interventions for eating disorders including binge eating. This systematic review and meta-analysis aimed to assess two decades of research on the efficacy of MBIs in reducing binge eating severity., Methods: We searched PubMed, Scopus and Cochrane Library for trials assessing the use of MBIs to treat binge eating severity in both clinical and non-clinical samples. The systematic review and meta-analysis was pre-registered at PROSPERO (CRD42020182395)., Results: Twenty studies involving 21 samples (11 RCT and 10 uncontrolled samples) met inclusion criteria. Random effects meta-analyses on the 11 RCT samples (n = 618: MBIs n = 335, controls n = 283) showed that MBIs significantly reduced binge eating severity (g = -0.39, 95% CI -0.68, -0.11) at end of trial, but was not maintained at follow-up (g = -0.06, 95% CI, -0.31, 0.20, k = 5). No evidence of publication bias was detected. On the Cochrane Risk of Bias Tool 2, trials were rarely rated at high risk of bias and drop-out rates did not differ between MBIs and control groups. MBIs also significantly reduced depression, and improved both emotion regulation and mindfulness ability., Conclusion: MBIs reduce binge eating severity at the end of trials. Benefits were not maintained at follow-up; however, only five studies were assessed. Future well-powered trials should focus on assessing diversity better, including more men and people from ethnic minority backgrounds., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by the wedge, REC1 domain and bridge helix (BH). We generated BH variants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity and mismatch sensitivity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. smFRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH variants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH variants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The lack of a nucleus is the defining cellular feature of bacteria and archaea. Consequently, transcription and translation are occurring in the same compartment, proceed simultaneously and likely in a coupled fashion. Recent cryo-electron microscopy (cryo-EM) and tomography data, also combined with crosslinking-mass spectrometry experiments, have uncovered detailed structural features of the coupling between a transcribing bacterial RNA polymerase (RNAP) and the trailing translating ribosome in Escherichia coli and Mycoplasma pneumoniae . Formation of this supercomplex, called expressome, is mediated by physical interactions between the RNAP-bound transcription elongation factors NusG and/or NusA and the ribosomal proteins including uS10. Based on the structural conservation of the RNAP core enzyme, the ribosome, and the universally conserved elongation factors Spt5 (NusG) and NusA, we discuss requirements and functional implications of transcription-translation coupling in archaea. We furthermore consider additional RNA-mediated and co-transcriptional processes that potentially influence expressome formation in archaea., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Weixlbaumer, Grünberger, Werner and Grohmann.)

Although copper is in many cases an essential micronutrient for cellular life, higher concentrations are toxic. Therefore, all living cells have developed strategies to maintain copper homeostasis. In this manuscript, we have analyzed the transcriptome-wide response of Pyrococcus furiosus to increased copper concentrations and described the essential role of the putative copper-sensing metalloregulator CopR in the detoxification process. To this end, we employed biochemical and biophysical methods to characterize the role of CopR. Additionally, a copR knockout strain revealed an amplified sensitivity in comparison to the parental strain towards increased copper levels, which designates an essential role of CopR for copper homeostasis. To learn more about the CopR-regulated gene network, we performed differential gene expression and ChIP-seq analysis under normal and 20 μM copper-shock conditions. By integrating the transcriptome and genome-wide binding data, we found that CopR binds to the upstream regions of many copper-induced genes. Negative-stain transmission electron microscopy and 2D class averaging revealed an octameric assembly formed from a tetramer of dimers for CopR, similar to published crystal structures from the Lrp family. In conclusion, we propose a model for CopR-regulated transcription and highlight the regulatory network that enables Pyrococcus to respond to increased copper concentrations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grünberger, Reichelt, Waege, Ned, Bronner, Kaljanac, Weber, El Ahmad, Knauss, Madej, Ziegler, Grohmann and Hausner.)

The DNA helicase Large helicase-related (Lhr) is present throughout archaea, including in the Asgard and Nanoarchaea, and has homologues in bacteria and eukaryotes. It is thought to function in DNA repair but in a context that is not known. Our data show that archaeal Lhr preferentially targets DNA replication fork structures. In a genetic assay, expression of archaeal Lhr gave a phenotype identical to the replication-coupled DNA repair enzymes Hel308 and RecQ. Purified archaeal Lhr preferentially unwound model forked DNA substrates compared with DNA duplexes, flaps and Holliday junctions, and unwound them with directionality. Single-molecule FRET measurements showed that binding of Lhr to a DNA fork causes ATP-independent distortion and base-pair melting at, or close to, the fork branchpoint. ATP-dependent directional translocation of Lhr resulted in fork DNA unwinding through the 'parental' DNA strands. Interaction of Lhr with replication forks in vivo and in vitro suggests that it contributes to DNA repair at stalled or broken DNA replication., (© 2020 The Author(s).)

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.

Urban parks are often designed and renewed using standardized models that fail to incorporate the different functions and services required by the local communities served. Furthermore, when the citizens are involved in co-design, the participation processes are seldom fully inclusive, and the results are not representative of the entire local community. The paper starts from these considerations for developing an innovative participatory design method to renew urban parks. This method simultaneously uses multiple and alternative participation tools for adapting to the different technical skills of the citizens and to their inclinations. Furthermore, it entails the analysis and interpretation of the expectations of the local community in light of the urban green network. The method uses the conceptual framework of the SWOT analysis and the Trochim's concept mapping for developing a card game playable face to face or online. The results of the card game are processed using the statistical analysis to identify the main design themes (the clusters), and to split the participants' suggestions into different design alternatives. Successively, the participants vote these alternatives for selecting the renewal design respondent to the local community's needs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The discovery of the archaeal domain of life is tightly connected to an in-depth analysis of the prokaryotic RNA world. In addition to Carl Woese's approach to use the sequence of the 16S rRNA gene as phylogenetic marker, the finding of Karl Stetter and Wolfram Zillig that archaeal RNA polymerases (RNAPs) were nothing like the bacterial RNAP but are more complex enzymes that resemble the eukaryotic RNAPII was one of the key findings supporting the idea that archaea constitute the third major branch on the tree of life. This breakthrough in transcriptional research 40years ago paved the way for in-depth studies of the transcription machinery in archaea. However, although the archaeal RNAP and the basal transcription factors that fine-tune the activity of the RNAP during the transcription cycle are long known, we still lack information concerning the architecture and dynamics of archaeal transcription complexes. In this context, single-molecule measurements were instrumental as they provided crucial insights into the process of transcription initiation, the architecture of the initiation complex and the dynamics of mobile elements of the RNAP. In this review, we discuss single-molecule approaches suitable to examine molecular mechanisms of transcription and highlight findings that shaped our understanding of the archaeal transcription apparatus. We furthermore explore the possibilities and challenges of next-generation single-molecule techniques, for example, super-resolution microscopy and single-molecule tracking, and ask whether these approaches will ultimately allow us to investigate archaeal transcription in vivo., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pyrococcus furiosus DSM 3638 is a model organism for hyperthermophilic archaea with an optimal growth temperature near 100°C. The genome was sequenced about 18 years ago. However, some publications suggest that in contrast to other Pyrococcus species, the genome of P. furiosus DSM 3638 is prone to genomic rearrangements. Therefore, we re-sequenced the genome using third generation sequencing techniques. The new de novo assembled genome is 1,889,914 bp in size and exhibits high sequence identity to the published sequence. However, two major deviations were detected: (1) The genome is 18,342 bp smaller than the NCBI reference genome due to a recently described deletion. (2) The region between PF0349 and PF0388 is inverted most likely due an assembly problem for the original sequence. In addition, numerous minor variations, ranging from single nucleotide exchanges, deletions or insertions were identified. The total number of insertion sequence (IS) elements is also reduced from 30 to 24 in the new sequence. Re-sequencing of a 2-year-old "lab culture" using Nanopore sequencing confirmed the overall stability of the P. furiosus DSM 3638 genome even under normal lab conditions without taking any special care. To improve genome annotation, the updated DNA sequence was combined with an RNA sequencing approach. Here, RNAs from eight different growth conditions were pooled to increase the number of detected transcripts. Furthermore, a differential RNA-Seq approach was employed for the identification of transcription start sites (TSSs). In total, 2515 TSSs were detected and classified into 834 primary (pTSS), 797 antisense (aTSS), 739 internal and 145 secondary TSSs. Our analysis of the upstream regions revealed a well conserved archaeal promoter structure. Interrogation of the distances between pTSSs and aTSSs revealed a significant number of antisense transcripts, which are a result of bidirectional transcription from the same TATA box. This mechanism of antisense transcript production could be further confirmed by in vitro transcription experiments. We assume that bidirectional transcription gives rise to non-functional antisense RNAs and that this is a widespread phenomenon in archaea due to the architecture of the TATA element and the symmetric structure of the TATA-binding protein.

In all domains of life, the regulation of transcription by DNA-dependent RNA polymerases (RNAPs) is achieved at the level of initiation to a large extent. Whereas bacterial promoters are recognized by a σ-factor bound to the RNAP, a complex set of transcription factors that recognize specific promoter elements is employed by archaeal and eukaryotic RNAPs. These initiation factors are of particular interest since the regulation of transcription critically relies on initiation rates and thus formation of pre-initiation complexes. The most conserved initiation factor is the TATA-binding protein (TBP), which is of crucial importance for all archaeal-eukaryotic transcription initiation complexes and the only factor required to achieve full rates of initiation in all three eukaryotic and the archaeal transcription systems. Recent structural, biochemical and genome-wide mapping data that focused on the archaeal and specialized RNAP I and III transcription system showed that the involvement and functional importance of TBP is divergent from the canonical role TBP plays in RNAP II transcription. Here, we review the role of TBP in the different transcription systems including a TBP-centric discussion of archaeal and eukaryotic initiation complexes. We furthermore highlight questions concerning the function of TBP that arise from these findings., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Sm-like (Lsm) proteins are found in all three domains of life. They are crucially involved in the RNA metabolism of prokaryotic organisms. To exert their function, they assemble into hexa- or heptameric rings and bind RNA via a conserved binding pocket for uridine stretches in the inner pore of the ring. Despite the conserved secondary structure of Lsm proteins, there are several features that lead to a structural diversification of this protein family that mediates their participation in a variety of processes related to RNA metabolism. Until recently, the cellular function of archaeal Sm-like proteins was not well understood. In this review, we discuss structural features of Lsm proteins with a strong focus on archaeal variants, reflect on the evolutionary development of archaeal Lsm proteins and present recent insights into their biological function., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Many biophysical techniques that are available to study the structure, function and dynamics of cellular constituents require modification of the target molecules. Site-specific labelling of a protein is of particular interest for fluorescence-based single-molecule measurements including single-molecule FRET or super-resolution microscopy. The labelling procedure should be highly specific but minimally invasive to preserve sensitive biomolecules. The modern molecular engineering toolkit provides elegant solutions to achieve the site-specific modification of a protein of interest often necessitating the incorporation of an unnatural amino acid to introduce a unique reactive moiety. The Amber suppression strategy allows the site-specific incorporation of unnatural amino acids into a protein of interest. Recently, this approach has been transferred to the mammalian expression system. Here, we demonstrate how the combination of unnatural amino acid incorporation paired with current bioorthogonal labelling strategies allow the site-specific engineering of fluorescent dyes into proteins produced in the cellular environment of a human cell. We describe in detail which parameters are important to ensure efficient incorporation of unnatural amino acids into a target protein in human expression systems. We furthermore outline purification and bioorthogonal labelling strategies that allow fast protein preparation and labelling of the modified protein. This way, the complete eukaryotic proteome becomes available for single-molecule fluorescence assays.

Transcription initiation by archaeal RNA polymerase (RNAP) and eukaryotic RNAP II requires the general transcription factor (TF) B/ IIB. Structural analyses of eukaryotic transcription initiation complexes locate the B-reader domain of TFIIB in close proximity to the active site of RNAP II. Here, we present the first crosslinking mapping data that describe the dynamic transitions of an archaeal TFB to provide evidence for structural rearrangements within the transcription complex during transition from initiation to early elongation phase of transcription. Using a highly specific UV-inducible crosslinking system based on the unnatural amino acid para-benzoyl-phenylalanine allowed us to analyze contacts of the Pyrococcus furiosus TFB B-reader domain with site-specific radiolabeled DNA templates in preinitiation and initially transcribing complexes. Crosslink reactions at different initiation steps demonstrate interactions of TFB with DNA at registers +6 to +14, and reduced contacts at +15, with structural transitions of the B-reader domain detected at register +10. Our data suggest that the B-reader domain of TFB interacts with nascent RNA at register +6 and +8 and it is displaced from the transcribed-strand during the transition from +9 to +10, followed by the collapse of the transcription bubble and release of TFB from register +15 onwards.

Fluorescence resonance energy transfer (FRET) has been instrumental in determining the structure and dynamics of biomolecules but distances above 8 nanometers are not accessible. However, with the advent and rapid development of super-resolution (SR) microscopy, distances between two fluorescent dyes below 20 nanometers can be resolved, which hitherto has been inaccessible for fluorescence microscopy approaches due to the limited resolving power of an optical imaging system that is determined by the fundamental laws of light diffraction (referred to as the diffraction limit). Therefore, the question arises whether SR microscopy can ultimately close the resolution gap between FRET and the diffraction limit and whether SR microscopy can be employed for the structural interrogation of proteins in the sub-20 nm range? Here, we show that the combination of DNA nanotechnology and single-molecule biochemistry allows the first step towards the investigation of the structural organization of a protein via SR microscopy. Limiting factors and possible future directions for the full implementation of SR microscopy as a structural tool are discussed.

Argonaute (Ago) proteins are encoded in all three domains of life and are responsible for the regulation of intracellular nucleic acid levels. Whereas some Ago variants are able to cleave target nucleic acids by their endonucleolytic activity, others only bind to their target nucleic acids while target cleavage is mediated by other effector proteins. Although all Ago proteins show a high degree of overall structural homology, the nature of the nucleic acid binding partners differs significantly. Recent structural and functional data have provided intriguing new insights into the mechanisms of archaeal and bacterial Ago variants demonstrating the mechanistic diversity within the prokaryotic Ago family with astonishing differences in nucleic acid selection and nuclease specificity. In this review, we provide an overview of the structural organisation of archaeal Ago variants and discuss the current understanding of their biological functions that differ significantly from their eukaryotic counterparts.

Human cells are complex entities in which molecular recognition and selection are critical for cellular processes often driven by structural changes and dynamic interactions. Biomolecules appear in different chemical states, and modifications, such as phosphorylation, affect their function. Hence, using proteins in their chemically native state in biochemical and biophysical assays is essential. Single-molecule FRET measurements allow exploration of the structure, function and dynamics of biomolecules but cannot be fully exploited for the human proteome, as a method for the site-specific coupling of organic dyes into native, non-recombinant mammalian proteins is lacking. We address this issue showing the site-specific engineering of fluorescent dyes into human proteins on the basis of bioorthogonal reactions. We show the applicability of the method to study functional and post-translationally modified proteins on the single-molecule level, among them the hitherto inaccessible human Argonaute 2., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Initiation of gene transcription by RNA polymerase (Pol) III requires the activity of TFIIIB, a complex formed by Brf1 (or Brf2), TBP (TATA-binding protein), and Bdp1. TFIIIB is required for recruitment of Pol III and to promote the transition from a closed to an open Pol III pre-initiation complex, a process dependent on the activity of the Bdp1 subunit. Here, we present a crystal structure of a Brf2-TBP-Bdp1 complex bound to DNA at 2.7 Å resolution, integrated with single-molecule FRET analysis and in vitro biochemical assays. Our study provides a structural insight on how Bdp1 is assembled into TFIIIB complexes, reveals structural and functional similarities between Bdp1 and Pol II factors TFIIA and TFIIF, and unravels essential interactions with DNA and with the upstream factor SNAPc. Furthermore, our data support the idea of a concerted mechanism involving TFIIIB and RNA polymerase III subunits for the closed to open pre-initiation complex transition.Transcription initiation by RNA polymerase III requires TFIIIB, a complex formed by Brf1/Brf2, TBP and Bdp1. Here, the authors describe the crystal structure of a Brf2-TBP-Bdp1 complex bound to a DNA promoter and characterize the role of Bdp1 in TFIIIB assembly and pre-initiation complex formation.

Transcription factors TBP and TF(II)B assemble with RNA polymerase at the promoter DNA forming the initiation complex. Despite a high degree of conservation, the molecular binding mechanisms of archaeal and eukaryotic TBP and TF(II)B differ significantly. Based on recent biophysical data, we speculate how the mechanisms co-evolved with transcription regulation and TBP multiplicity.

Argonaute (Ago) proteins in eukaryotes are known as key players in post-transcriptional gene silencing 1 , while recent studies on prokaryotic Agos hint at their role in the protection against invading DNA 2,3 . Here, we present crystal structures of the apo enzyme and a binary Ago-guide complex of the archaeal Methanocaldococcus jannaschii (Mj) Ago. Binding of a guide DNA leads to large structural rearrangements. This includes the structural transformation of a hinge region containing a switch helix, which has been shown for human Ago2 to be critical for the dynamic target search process 4-6 . To identify key residues crucial for MjAgo function, we analysed the effect of several MjAgo mutants. We observe that the nature of the 3' and 5' nucleotides in particular, as well as the switch helix, appear to impact MjAgo cleavage activity. In summary, we provide insights into the molecular mechanisms that drive DNA-guided DNA silencing by an archaeal Ago.

Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates in a guide-independent fashion primes MjAgo for subsequent rounds of DNA cleavage. Chromatinized genomic DNA is resistant to MjAgo degradation, and recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows that key residues important for guide-dependent target processing are also involved in guide-independent MjAgo function. This is the first characterization of guide-independent cleavage activity for an Argonaute protein potentially serving as a guide biogenesis pathway in a prokaryotic system.

Site-directed spin labeling for EPR- and NMR spectroscopy has mainly been achieved exploiting the specific reactivity of cysteines. For proteins with native cysteines or for in vivo applications, an alternative coupling strategy is required. In these cases click chemistry offers major benefits by providing a fast and highly selective, biocompatible reaction between azide and alkyne groups. Here, we establish click chemistry as a tool to target unnatural amino acids in vitro and in vivo using azide- and alkyne-functionalized spin labels. The approach is compatible with a variety of labels including reduction-sensitive nitroxides. Comparing spin labeling efficiencies from the copper-free with the strongly reducing copper(I)-catalyzed azide-alkyne click reaction, we find that the faster kinetics for the catalyzed reaction outrun reduction of the labile nitroxide spin labels and allow quantitative labeling yields within short reaction times. Inter-spin distance measurements demonstrate that the novel side chain is suitable for paramagnetic NMR- or EPR-based conformational studies of macromolecular complexes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In all living organisms, the flow of genetic information is a two-step process: first DNA is transcribed into RNA, which is subsequently used as template for protein synthesis during translation. In bacteria, archaea and eukaryotes, transcription is carried out by multi-subunit RNA polymerases (RNAPs) sharing a conserved architecture of the RNAP core. RNAPs catalyse the highly accurate polymerisation of RNA from NTP building blocks, utilising DNA as template, being assisted by transcription factors during the initiation, elongation and termination phase of transcription. The complexity of this highly dynamic process is reflected in the intricate network of protein-protein and protein-nucleic acid interactions in transcription complexes and the substantial conformational changes of the RNAP as it progresses through the transcription cycle.In this chapter, we will first briefly describe the early work that led to the discovery of multisubunit RNAPs. We will then discuss the three-dimensional organisation of RNAPs from the bacterial, archaeal and eukaryotic domains of life, highlighting the conserved nature, but also the domain-specific features of the transcriptional apparatus. Another section will focus on transcription factors and their role in regulating the RNA polymerase throughout the different phases of the transcription cycle. This includes a discussion of the molecular mechanisms and dynamic events that govern transcription initiation, elongation and termination.

Deciphering the molecular mechanisms of eukaryotic Argonaute proteins is crucial for the understanding of RNA interference (RNAi), a posttranscriptional gene silencing process. Fluorescence-based single-molecule studies like single-molecule Förster resonance energy transfer (FRET) between a donor and acceptor dye represent a versatile tool to gain a mechanistic understanding of the structural dynamics of a biomolecular complex. Until today it was not possible to site-specifically introduce fluorophores into eukaryotic Argonaute. Using an archaeal Argonaute variant from Methanocaldococcus jannaschii that closely resembles its eukaryotic counterpart, we site-specifically incorporated fluorescent probes into Argonaute. In this chapter, we first describe how to express archaeal Argonaute with the site-specifically engineered unnatural amino acid para-azido-L-phenylalanine (pAzF) and subsequently describe the coupling of a fluorophore exploiting the unique chemistry of the azide group of pAzF. In the second part of the chapter, we present a methodological approach that probes complex formation between acceptor-labeled archaeal Argonaute and guide and target nucleic acids equipped with a donor fluorophore which ultimately allows single-molecule FRET measurements. Furthermore we describe binding and cleavage assays that report on the functionality of Argonaute-nucleic acid complexes.

Little is known about how archaeal viruses perturb the transcription machinery of their hosts. Here we provide the first example of an archaeo-viral transcription factor that directly targets the host RNA polymerase (RNAP) and efficiently represses its activity. ORF145 from the temperate Acidianus two-tailed virus (ATV) forms a high-affinity complex with RNAP by binding inside the DNA-binding channel where it locks the flexible RNAP clamp in one position. This counteracts the formation of transcription pre-initiation complexes in vitro and represses abortive and productive transcription initiation, as well as elongation. Both host and viral promoters are subjected to ORF145 repression. Thus, ORF145 has the properties of a global transcription repressor and its overexpression is toxic for Sulfolobus. On the basis of its properties, we have re-named ORF145 RNAP Inhibitory Protein (RIP).

Forces in biological systems are typically investigated at the single-molecule level with atomic force microscopy or optical and magnetic tweezers, but these techniques suffer from limited data throughput and their requirement for a physical connection to the macroscopic world. We introduce a self-assembled nanoscopic force clamp built from DNA that operates autonomously and allows massive parallelization. Single-stranded DNA sections of an origami structure acted as entropic springs and exerted controlled tension in the low piconewton range on a molecular system, whose conformational transitions were monitored by single-molecule Förster resonance energy transfer. We used the conformer switching of a Holliday junction as a benchmark and studied the TATA-binding protein-induced bending of a DNA duplex under tension. The observed suppression of bending above 10 piconewtons provides further evidence of mechanosensitivity in gene regulation., (Copyright © 2016, American Association for the Advancement of Science.)

Argonaute (Ago) proteins from all three domains of life are key players in processes that specifically regulate cellular nucleic acid levels. Some of these Ago proteins, among them human Argonaute2 (hAgo2) and Ago from the archaeal organism Methanocaldococcus jannaschii (MjAgo), are able to cleave nucleic acid target strands that are recognised via an Ago-associated complementary guide strand. Here we present an in-depth kinetic side-by-side analysis of hAgo2 and MjAgo guide and target substrate binding as well as target strand cleavage, which enabled us to disclose similarities and differences in the mechanistic pathways as a function of the chemical nature of the substrate. Testing all possible guide-target combinations (i.e. RNA/RNA, RNA/DNA, DNA/RNA and DNA/DNA) with both Ago variants we demonstrate that the molecular mechanism of substrate association is highly conserved among archaeal-eukaryotic Argonautes. Furthermore, we show that hAgo2 binds RNA and DNA guide strands in the same fashion. On the other hand, despite striking homology between the two Ago variants, MjAgo cannot orientate guide RNA substrates in a way that allows interaction with the target DNA in a cleavage-compatible orientation., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2016. Published by Elsevier Ltd.)

For single-molecule localization based superresolution, the concentration of fluorescent labels has to be thinned out. This is commonly achieved by photophysically or photochemically deactivating subsets of molecules. Alternatively, apparent switching of molecules can be achieved by transient binding of fluorescent labels. Here, a diffusing dye yields bright fluorescent spots when binding to the structure of interest. As the binding interaction is weak, the labeling is reversible and the dye ligand construct diffuses back into solution. This approach of achieving superresolution by transient binding (STB) is reviewed in this manuscript. Different realizations of STB are discussed and compared to other localization-based superresolution modalities. We propose the development of labeling strategies that will make STB a highly versatile tool for superresolution microscopy at highest resolution., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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.