The NEXT-White detector, a high-pressure gaseous xenon time projection chamber, demonstrated the excellence of this technology for future neutrinoless double beta decay searches using photomultiplier ...tubes (PMTs) to measure energy and silicon photomultipliers (SiPMs) to extract topology information. This analysis uses \(^{83m}\text{Kr}\) data from the NEXT-White detector to measure and understand the energy resolution that can be obtained with the SiPMs, rather than with PMTs. The energy resolution obtained of (10.9 \(\pm\) 0.6) \(\%\), full-width half-maximum, is slightly larger than predicted based on the photon statistics resulting from very low light detection coverage of the SiPM plane in the NEXT-White detector. The difference in the predicted and measured resolution is attributed to poor corrections, which are expected to be improved with larger statistics. Furthermore, the noise of the SiPMs is shown to not be a dominant factor in the energy resolution and may be negligible when noise subtraction is applied appropriately, for high-energy events or larger SiPM coverage detectors. These results, which are extrapolated to estimate the response of large coverage SiPM planes, are promising for the development of future, SiPM-only, readout planes that can offer imaging and achieve similar energy resolution to that previously demonstrated with PMTs.
The imaging of individual Ba\(^{2+}\) ions in high pressure xenon gas is one possible way to attain background-free sensitivity to neutrinoless double beta decay and hence establish the Majorana ...nature of the neutrino. In this paper we demonstrate selective single Ba\(^{2+}\) ion imaging inside a high-pressure xenon gas environment. Ba\(^{2+}\) ions chelated with molecular chemosensors are resolved at the gas-solid interface using a diffraction-limited imaging system with scan area of 1\(\times\)1~cm\(^2\) located inside 10~bar of xenon gas. This new form of microscopy represents an important enabling step in the development of barium tagging for neutrinoless double beta decay searches in \(^{136}\)Xe, as well as a new tool for studying the photophysics of fluorescent molecules and chemosensors at the solid-gas interface.
NEXT-100 is currently being constructed at the Laboratorio Subterráneo de Canfranc in the Spanish Pyrenees and will search for neutrinoless double beta decay using a high-pressure gaseous time ...projection chamber (TPC) with 100 kg of xenon. Charge amplification is carried out via electroluminescence (EL) which is the process of accelerating electrons in a high electric field region causing secondary scintillation of the medium proportional to the initial charge. The NEXT-100 EL and cathode regions are made from tensioned hexagonal meshes of 1 m diameter. This paper describes the design, characterization, and installation of these parts for NEXT-100. Simulations of the electric field are performed to model the drift and amplification of ionization electrons produced in the detector under various EL region alignments and rotations. Measurements of the electrostatic breakdown voltage in air characterize performance under high voltage conditions and identify breakdown points. The electrostatic deflection of the mesh is quantified and fit to a first-principles mechanical model. Measurements were performed with both a standalone test EL region and with the NEXT-100 EL region before its installation in the detector. Finally, we describe the parts as installed in NEXT-100, following their deployment in Summer 2023.
Noble element time projection chambers are a leading technology for rare event detection in physics, such as for dark matter and neutrinoless double beta decay searches. Time projection chambers ...typically assign event position in the drift direction using the relative timing of prompt scintillation and delayed charge collection signals, allowing for reconstruction of an absolute position in the drift direction. In this paper, alternate methods for assigning event drift distance via quantification of electron diffusion in a pure high pressure xenon gas time projection chamber are explored. Data from the NEXT-White detector demonstrate the ability to achieve good position assignment accuracy for both high- and low-energy events. Using point-like energy deposits from \(^{83\mathrm{m}}\)Kr calibration electron captures (\(E\sim45\)keV), the position of origin of low-energy events is determined to \(2~\)cm precision with bias \(< 1\)mm. A convolutional neural network approach is then used to quantify diffusion for longer tracks (E\(\geq\)1.5MeV), yielding a precision of 3cm on the event barycenter. The precision achieved with these methods indicates the feasibility energy calibrations of better than 1% FWHM at Q\(_{\beta\beta}\) in pure xenon, as well as the potential for event fiducialization in large future detectors using an alternate method that does not rely on primary scintillation.
All cellular genetic information is transcribed into RNA by multisubunit RNA polymerases (RNAPs). The basal transcription initiation factors of cellular RNAPs stimulate the initial RNA synthesis via ...poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The nucleotide at the RNA 3' end determines the pause lifetime. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The antipausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This “RNAP boosting” activity of the initiation factor is shaped by the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.
Transcription initiation is a key checkpoint and highly regulated step of gene expression. The sigma (σ) subunit of RNA polymerase (RNAP) controls all transcription initiation steps, from recognition ...of the −10/−35 promoter elements, upon formation of the closed promoter complex (RPc), to stabilization of the open promoter complex (RPo) and stimulation of the primary steps in RNA synthesis. The canonical mechanism to regulate σ activity upon transcription initiation relies on activators that recognize specific DNA motifs and recruit RNAP to promoters. This mini-review describes an emerging group of transcriptional regulators that form a complex with σ or/and RNAP prior to promoter binding, remodel the σ subunit conformation, and thus modify RNAP activity. Such strategy is widely used by bacteriophages to appropriate the host RNAP. Recent findings on RNAP-binding protein A (RbpA) from
Mycobacterium tuberculosis
and Crl from
Escherichia coli
suggest that activator-driven changes in σ conformation can be a widespread regulatory mechanism in bacteria.
Initiation of RNA synthesis by bacterial RNA polymerase (RNAP) requires melting of promoter DNA, which is nucleated by the σ subunit during formation of the “open” promoter complex (RPo). The ...antibiotic lipiarmycin (Lpm) inhibits promoter melting by blocking access of the template DNA strand to the RNAP active-site cleft. Here we show that Escherichia coli RNAP holoenzymes containing either housekeeping σ70, with a deletion in the region 3.2, or the stationary phase σS subunits exhibited hypersensitivity to Lpm and increased cold sensitivity of RPo formation. Similar effects were produced by mutation located ~60Å away from the Lpm binding site within σ70 region 1.2, controlling −10 promoter element recognition. Our data suggested that template strand single-stranded DNA competes with Lpm for binding to RNAP and that σ70 regions 1.2 and 3.2 attenuate Lpm action by promoting DNA duplex opening.
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•Promoter DNA opening is a target for genetic regulation and for antibiotics.•Regions of the σ70 factor implicated in DNA opening antagonize the lipiarmycin action.•RNAP containing the stationary phase σS factor displayed hypersensitivity to lipiarmycin.•Antibiotic can function as the σ factor selective transcriptional repressor.
In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial ...transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause (“initiation pause,” ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.
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•E. coli RNA polymerase pauses during initial transcription at lac promoters•Initiation pausing lasts for ∼20 s and occurs at the transition from 6- to 7-nt RNA•Region 3.2 of σ70 is the main protein element controlling pausing•Pausing is likely to be controlled further by a complex set of determinants
Initial transcription involves synthesis of short RNAs that are either released or extended to allow promoter escape; such mechanisms are unclear due to heterogeneity. Duchi et al. used single-molecule fluorescence to visualize transcription in real time and discovered a long pause after synthesis of a 6-mer RNA.
All cellular genetic information is transcribed into RNA by multisubunit RNA polymerases (RNAPs). The basal transcription initiation factors of cellular RNAPs stimulate the initial RNA synthesis via ...poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The nucleotide at the RNA 3' end determines the pause lifetime. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The anti-pausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This "RNAP boosting" activity of the initiation factor is shaped by the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.
Self-assembly of macromolecules into higher-order symmetric structures is fundamental for the regulation of biological processes. Higher-order symmetric structure self-assembly by the gene expression ...machinery, such as bacterial DNA-dependent RNA polymerase (RNAP), has never been reported before. Here, we show that the stress-response σ
factor from the human pathogen, Mycobacterium tuberculosis, induces the RNAP holoenzyme oligomerization into a supramolecular complex composed of eight RNAP units. Cryo-electron microscopy revealed a pseudo-symmetric structure of the RNAP octamer in which RNAP protomers are captured in an auto-inhibited state and display an open-clamp conformation. The structure shows that σ
is sequestered by the RNAP flap and clamp domains. The transcriptional activator RbpA prevented octamer formation by promoting the initiation-competent RNAP conformation. Our results reveal that a non-conserved region of σ is an allosteric controller of transcription initiation and demonstrate how basal transcription factors can regulate gene expression by modulating the RNAP holoenzyme assembly and hibernation.
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