313 research outputs found
Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release.
Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons
Dopamine D1 vs D5 receptor-dependent induction of seizures in relation to DARPP-32, ERK1/2 and GluR1-AMPA signalling.
Recent reports have shown that the selective dopamine D(1)-like agonist SKF 83822 [which stimulates adenylate cyclase, but not phospholipase C] induces prominent behavioral seizures in mice, whereas its benzazepine congener SKF 83959 [which stimulates phospholipase C, but not adenylate cyclase] does not. To investigate the relative involvement of D(1) vs D(5) receptors in mediating seizures, ethological behavioral topography and cortical EEGs were recorded in D(1), D(5) and DARPP-32 knockout mice in response to a convulsant dose of SKF 83822. SKF 83822-induced behavioral and EEG seizures were gene dose-dependently abolished in D(1) knockouts. In both heterozygous and homozygous D(5) knockouts, the latency to first seizure was significantly increased and total EEG seizures were reduced relative to wild-types. The majority (60%) of homozygous DARPP-32 knockouts did not have seizures; of those having seizures (40%), the latency to first seizure was significantly increased and the number of high amplitude, high frequency polyspike EEG events was reduced. In addition, immunoblotting was performed to investigate downstream intracellular signalling mechanisms at D(1)-like receptors following challenge with SKF 83822 and SKF 83959. In wild-types administered SKF 83822, levels of ERK1/2 and GluR1 AMPA receptor phosphorylation increased two-fold in both the striatum and hippocampus; in striatal slices DARPP-32 phosphorylation at Thr34 increased five-fold relative to vehicle-treated controls. These findings indicate that D(1), and to a lesser extent D(5), receptor coupling to DARPP-32, ERK1/2 and glutamatergic signalling is involved in mediating the convulsant effects of SKF 83822
Paleocene–Eocene age glendonites from the Mid-Norwegian Margin – indicators of cold snaps in the hothouse?
The International Ocean Discovery Program (IODP) Expedition 396 to the mid-Norwegian margin recovered > 1300 m of pristinely preserved, volcanic-ash-rich sediments deposited during the late Paleocene and early Eocene from close to the centre of the North Atlantic Igneous Province (NAIP). Remarkably, many of these cores contain glendonites, pseudomorphs after the purported cold-water mineral ikaite, from sediments dated to the late Paleocene and early Eocene. These time intervals span some of the hottest climates of the Cenozoic, including the Paleocene–Eocene Thermal Maximum (PETM). Global deep-ocean temperatures are not thought to have dropped below 10 ∘C at any point during this time, making the occurrence of supposedly cold-water (near-freezing temperature) glendonite pseudomorphs seemingly paradoxical. This study presents a detailed sedimentological, geochemical, and microscopic study of the IODP Exp. 396 glendonites and presents an updated model for the ikaite-to-calcite transformation for these glendonites. Specifically, we show that early diagenesis of basaltic ashes of the NAIP appear to have chemically promoted ikaite growth in the sediments in this region. Together with existing knowledge of late Paleocene and early Eocene glendonites from Svalbard to the north and early Eocene glendonites from Denmark to the south, these new glendonite finds possibly imply episodic, short-duration, and likely localized cooling in the Nordic Seas region, which may have been directly or indirectly linked to the emplacement of the NAIP
Characterisation of the contribution of the GABA-benzodiazepine α1 receptor subtype to [11C]Ro15-4513 PET images
This positron emission tomography (PET) study aimed to further define selectivity of [11C]Ro15-4513 binding to the GABARα5 relative to the GABARα1 benzodiazepine receptor subtype. The impact of zolpidem, a GABARα1-selective agonist, on [11C]Ro15-4513, which shows selectivity for GABARα5, and the nonselective benzodiazepine ligand [11C]flumazenil binding was assessed in humans. Compartmental modelling of the kinetics of [11C]Ro15-4513 time-activity curves was used to describe distribution volume (VT) differences in regions populated by different GABA receptor subtypes. Those with low α5 were best fitted by one-tissue compartment models; and those with high α5 required a more complex model. The heterogeneity between brain regions suggested spectral analysis as a more appropriate method to quantify binding as it does not a priori specify compartments. Spectral analysis revealed that zolpidem caused a significant VT decrease (∼10%) in [11C]flumazenil, but no decrease in [11C]Ro15-4513 binding. Further analysis of [11C]Ro15-4513 kinetics revealed additional frequency components present in regions containing both α1 and α5 subtypes compared with those containing only α1. Zolpidem reduced one component (mean±s.d.: 71%±41%), presumed to reflect α1-subtype binding, but not another (13%±22%), presumed to reflect α5. The proposed method for [11C]Ro15-4513 analysis may allow more accurate selective binding assays and estimation of drug occupancy for other nonselective ligands
Nanopore ReCappable sequencing maps SARS-CoV-2 5′ capping sites and provides new insights into the structure of sgRNAs
The SARS-CoV-2 virus has a complex transcriptome characterised by multiple, nested subgenomic RNAsused to express structural and accessory proteins. Long-read sequencing technologies such as nanopore direct RNA sequencing can recover full-length transcripts, greatly simplifying the assembly of structurally complex RNAs. However, these techniques do not detect the 5 ' cap, thus preventing reliable identification and quantification of full-length, coding transcript models. Here we used Nanopore ReCappable Sequencing (NRCeq), a new technique that can identify capped full-length RNAs, to assemble a complete annotation of SARS-CoV-2 sgRNAs and annotate the location of capping sites across the viral genome. We obtained robust estimates of sgRNA expression across cell lines and viral isolates and identified novel canonical and non-canonical sgRNAs, including one that uses a previously un-annotated leader-to-body junction site. The data generated in this work constitute a useful resource for the scientific community and provide important insights into the mechanisms that regulate the transcription of SARS-CoV-2 sgRNAs
A Self-Consistent Model for Positronium Formation from Helium Atoms
The differential and total cross sections for electron capture by positrons
from helium atoms are calculated using a first-order distorted wave theory
satisfying the Coulomb boundary conditions. In this formalism a parametric
potential is used to describe the electron screening in a consistent and
realistic manner. The present procedure is self consistent because (i) it
satisfies the correct boundary conditions and post-prior symmetry, and (ii) the
potential and the electron binding energies appearing in the transition
amplitude are consistent with the wave functions describing the collision
system. The results are compared with the other theories and with the available
experimental measurements. At the considered range of collision energies, the
results agree reasonably well with recent experiments and theories.
[Note: This paper will be published on volume 42 of the Brazilian Journal of
Physics
Altered Backbone and Side-Chain Interactions Result in Route Heterogeneity during the Folding of Interleukin-1b (IL-1b)
Deletion of the b-bulge trigger-loop results in both a switch in the preferred folding route, from the functional loop
packing folding route to barrel closure, as well as conversion of the agonist activity of IL-1b into antagonist activity. Conversely,
circular permutations of IL-1b conserve the functional folding route as well as the agonist activity. These two extremes in the
folding-functional interplay beg the question of whether mutations in IL-1b would result in changes in the populations of heterogeneous
folding routes and the signaling activity. A series of topologically equivalent water-mediated b-strand bridging interactions
within the pseudosymmetric b-trefoil fold of IL-1b highlight the backbone water interactions that stabilize the secondary and
tertiary structure of the protein. Additionally, conserved aromatic residues lining the central cavity appear to be essential for both
stability and folding. Here, we probe these protein backbone-water molecule and side chain-side chain interactions and the role
they play in the folding mechanism of this geometrically stressed molecule. We used folding simulations with structure-based
models, as well as a series of folding kinetic experiments to examine the effects of the F42W core mutation on the folding landscape
of IL-1b. This mutation alters water-mediated backbone interactions essential for maintaining the trefoil fold. Our results
clearly indicate that this perturbation in the primary structure alters a structural water interaction and consequently modulates the
population of folding routes accessed during folding and signaling activity
The Energy Landscape, Folding Pathways and the Kinetics of a Knotted Protein
The folding pathway and rate coefficients of the folding of a knotted protein
are calculated for a potential energy function with minimal energetic
frustration. A kinetic transition network is constructed using the discrete
path sampling approach, and the resulting potential energy surface is
visualized by constructing disconnectivity graphs. Owing to topological
constraints, the low-lying portion of the landscape consists of three distinct
regions, corresponding to the native knotted state and to configurations where
either the N- or C-terminus is not yet folded into the knot. The fastest
folding pathways from denatured states exhibit early formation of the
N-terminus portion of the knot and a rate-determining step where the C-terminus
is incorporated. The low-lying minima with the N-terminus knotted and the
C-terminus free therefore constitute an off-pathway intermediate for this
model. The insertion of both the N- and C-termini into the knot occur late in
the folding process, creating large energy barriers that are the rate limiting
steps in the folding process. When compared to other protein folding proteins
of a similar length, this system folds over six orders of magnitude more
slowly.Comment: 19 page
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TAO Conceptual Design Report: A Precision Measurement of the Reactor Antineutrino Spectrum with Sub-percent Energy Resolution
The Taishan Antineutrino Observatory (TAO, also known as JUNO-TAO) is a
satellite experiment of the Jiangmen Underground Neutrino Observatory (JUNO). A
ton-level liquid scintillator detector will be placed at about 30 m from a core
of the Taishan Nuclear Power Plant. The reactor antineutrino spectrum will be
measured with sub-percent energy resolution, to provide a reference spectrum
for future reactor neutrino experiments, and to provide a benchmark measurement
to test nuclear databases. A spherical acrylic vessel containing 2.8 ton
gadolinium-doped liquid scintillator will be viewed by 10 m^2 Silicon
Photomultipliers (SiPMs) of >50% photon detection efficiency with almost full
coverage. The photoelectron yield is about 4500 per MeV, an order higher than
any existing large-scale liquid scintillator detectors. The detector operates
at -50 degree C to lower the dark noise of SiPMs to an acceptable level. The
detector will measure about 2000 reactor antineutrinos per day, and is designed
to be well shielded from cosmogenic backgrounds and ambient radioactivities to
have about 10% background-to-signal ratio. The experiment is expected to start
operation in 2022
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