15 research outputs found
Characterization of Transcription from TATA-Less Promoters: Identification of a New Core Promoter Element XCPE2 and Analysis of Factor Requirements
More than 80% of mammalian protein-coding genes are driven by TATA-less promoters which often show multiple transcriptional start sites (TSSs). However, little is known about the core promoter DNA sequences or mechanisms of transcriptional initiation for this class of promoters.Here we identify a new core promoter element XCPE2 (X core promoter element 2) (consensus sequence: A/C/G-C-C/T-C-G/A-T-T-G/A-C-C/A(+1)-C/T) that can direct specific transcription from the second TSS of hepatitis B virus X gene mRNA. XCPE2 sequences can also be found in human promoter regions and typically appear to drive one of the start sites within multiple TSS-containing TATA-less promoters. To gain insight into mechanisms of transcriptional initiation from this class of promoters, we examined requirements of several general transcription factors by in vitro transcription experiments using immunodepleted nuclear extracts and purified factors. Our results show that XCPE2-driven transcription uses at least TFIIB, either TFIID or free TBP, RNA polymerase II (RNA pol II) and the MED26-containing mediator complex but not Gcn5. Therefore, XCPE2-driven transcription can be carried out by a mechanism which differs from previously described TAF-dependent mechanisms for initiator (Inr)- or downstream promoter element (DPE)-containing promoters, the TBP- and SAGA (Spt-Ada-Gcn5-acetyltransferase)-dependent mechanism for yeast TATA-containing promoters, or the TFTC (TBP-free-TAF-containing complex)-dependent mechanism for certain Inr-containing TATA-less promoters. EMSA assays using XCPE2 promoter and purified factors further suggest that XCPE2 promoter recognition requires a set of factors different from those for TATA box, Inr, or DPE promoter recognition.We identified a new core promoter element XCPE2 that are found in multiple TSS-containing TATA-less promoters. Mechanisms of promoter recognition and transcriptional initiation for XCPE2-driven promoters appear different from previously shown mechanisms for classical promoters that show single "focused" TSSs. Our studies provide insight into novel mechanisms of RNA Pol II transcription from multiple TSS-containing TATA-less promoters
Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries
Abstract
Background
Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres.
Methods
This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries.
Results
In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia.
Conclusion
This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries
Evaluation of non-invasive genetic sampling methods for estimating tiger population size
There is often a conservation need to estimate population abundances of elusive, low-density, wide-ranging carnivore species. Because of logistical constraints, investigators often employ non-invasive 'captures' that may involve 'genetic' or 'photographic' sampling in such cases. Established capture-recapture (CR) methods offer a powerful analytical tool for such data. In this paper, we developed a rigorous combination of captive, laboratory and field-based protocols for identifying individual tigers (Panthera tigris) from fecal DNA. We explored trade-offs between numbers of microsatellite loci used for reliable individual identifications and the need for higher capture rates for robust analyses. Our field surveys of scats were also specifically designed for CR analyses, enabling us to test for population closure, estimate capture probabilities and tiger abundance. Consequently, we could compare genetic capture estimates to results of a 'photographic capture' study of tigers at the same site. The estimates using the heterogeneity model (Mh-Jackknife) for fecal DNA survey were [Mt+1 = 26; P^_ = 0.09 and N^(SE^[N^]) =66 (12.98)] in close agreement with those from the photographic survey [(Mt+1 = 29; P^_ = 0.04 and N^(SE^[N^]) = 66 (13.8)]. Our results revealed that designing field surveys of scats explicitly for CR data analyses generate reliable estimates of capture probability and abundance for elusive, low density species such as tigers. The study also highlights the importance of rigorous field survey and laboratory protocols for reliable abundance estimation in contexts where other approaches such as camera-trapping or physical tagging of animals may not be practical options
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Phosphorylation cascade regulates the formation and maturation of rotaviral replication factories.
The rotavirus (RV) genome is replicated and packaged into virus progeny in cytoplasmic inclusions called viroplasms, which require interactions between RV nonstructural proteins NSP2 and NSP5. How viroplasms form remains unknown. We previously found two forms of NSP2 in RV-infected cells: a cytoplasmically dispersed dNSP2, which interacts with hypophosphorylated NSP5; and a viroplasm-specific vNSP2, which interacts with hyperphosphorylated NSP5. Other studies report that CK1α, a ubiquitous cellular kinase, hyperphosphorylates NSP5, but requires NSP2 for reasons that are unclear. Here we show that silencing CK1α in cells before RV infection resulted in (i) >90% decrease in RV replication, (ii) disrupted vNSP2 and NSP5 interaction, (iii) dispersion of vNSP2 throughout the cytoplasm, and (iv) reduced vNSP2 protein levels. Together, these data indicate that CK1α directly affects NSP2. Accordingly, an in vitro kinase assay showed that CK1α phosphorylates serine 313 of NSP2 and triggers NSP2 octamers to form a lattice structure as demonstrated by crystallographic analysis. Additionally, a dual-specificity autokinase activity for NSP2 was identified and confirmed by mass spectrometry. Together, our studies show that phosphorylation of NSP2 involving CK1α controls viroplasm assembly. Considering that CK1α plays a role in the replication of other RNA viruses, similar phosphorylation-dependent mechanisms may exist for other virus pathogens that require cytoplasmic virus factories for replication
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2.7 Å cryo-EM structure of rotavirus core protein VP3, a unique capping machine with a helicase activity.
In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 caps the nascent transcripts synthesized from the genomic dsRNA segments by the RV polymerase VP1 within the particle core. Here, from cryo-electron microscopy, x-ray crystallography, and biochemical analyses, we show that VP3 forms a stable tetrameric assembly with each subunit having a modular domain organization, which uniquely integrates five distinct enzymatic steps required for capping the transcripts. In addition to the previously known guanylyl- and methyltransferase activities, we show that VP3 exhibits hitherto unsuspected RNA triphosphatase activity necessary for initiating transcript capping and RNA helicase activity likely required for separating the RNA duplex formed transiently during endogenous transcription. From our studies, we propose a new mechanism for how VP3 inside the virion core caps the nascent transcripts exiting from the polymerase
Poly(ADP-ribose) Polymerase 1 Interacts with Nuclear Respiratory Factor 1 (NRF-1) and Plays a Role in NRF-1 Transcriptional Regulation*S⃞
Nuclear respiratory factor 1 (NRF-1) is one of the key transcriptional
activators for nuclear-coded genes involved in mitochondrial biogenesis and
function as well as for many housekeeping genes. A transcriptional
co-activator PGC-1 and its related family member PRC have previously been
shown to interact with NRF-1 and co-activate NRF-1. We show here that NRF-1
can also directly interact with poly(ADP-ribose) polymerase 1 (PARP-1) and
co-purify the
PARP-1·DNA-PK·Ku80·Ku70·topoisomerase
IIβ-containing protein complex. Our in vitro binding experiments
show that DNA-binding/dimerization domain of NRF-1 and the N-terminal half of
PARP-1, which contains two Zinc fingers and the auto-modification domain, are
responsible for the interaction, and that this interaction occurs with or
without PARP-1 poly(ADP-ribosyl)ation (PARylation). DNA-bound NRF-1 can form a
complex with PARP-1, suggesting that NRF-1 can recruit the
PARP-1·DNA-PK·Ku80·Ku70·topoisomerase
IIβ-containing protein complex to the promoter. PARP-1 can also PARylate
the DNA-binding domain of NRF-1 and negatively regulate NRF-1·PARP-1
interaction. Transient transfection and chromatin immunoprecipitation
experiments suggest that PARP-1 plays a role during transcriptional activation
by NRF-1. Our finding identifies a new aspect of transcriptional regulation
used by NRF-1