Ribosome*RelA structures reveal the mechanism of stringent response activation

Stringent response is a conserved bacterial stress response underlying virulence and antibiotic resistance. RelA/SpoT-homolog proteins synthesize transcriptional modulators (p)ppGpp, allowing bacteria to adapt to stress. RelA is activated during amino-acid starvation, when cognate deacyl-tRNA binds to the ribosomal A (aminoacyl-tRNA) site. We report four cryo-EM structures of E. coli RelA bound to the 70S ribosome, in the absence and presence of deacyl-tRNA accommodating in the 30S A site. The boomerang-shaped RelA with a wingspan of more than 100 A wraps around the A/R (30S A-site/RelA-bound) tRNA. The CCA end of the A/R tRNA pins the central TGS domain against the 30S subunit, presenting the (p)ppGpp-synthetase domain near the 30S spur. The ribosome and A/R tRNA are captured in three conformations, revealing hitherto elusive states of tRNA engagement with the ribosomal decoding center. Decoding-center rearrangements are coupled with the step-wise 30S-subunit \u27closure\u27, providing insights into the dynamics of high-fidelity tRNA decoding

TRAINING OF PEDAGOGICAL EDUCATION MASTERS: PRACTICE-ORIENTED MODEL

Purpose: The article deals with the problems of training, attraction, and retention of teachers of the required qualification in educational institutions. Methodology: This problem is analyzed at the regional level in the context of reforms in the higher education system, the transition to a two-level model in accordance with the provisions of the Bologna process. Result: Based on the analysis of open data in the work it is stated that the shortage of teachers in the region (Samara region) is significant and has a negative impact on the educational process of educational institutions; it cannot be compensated exclusively by bachelors of pedagogical education, issued by pedagogical universities. Applications: This research can be used for universities, teachers, and students. Novelty/Originality: In this research, the model of training of pedagogical education masters: practice-oriented model is presented in a comprehensive and complete manner

Structures of Yeast 80S Ribosome-tRNA Complexes in the Rotated and Nonrotated Conformations

SummaryThe structural understanding of eukaryotic translation lags behind that of translation on bacterial ribosomes. Here, we present two subnanometer resolution structures of S. cerevisiae 80S ribosome complexes formed with either one or two tRNAs and bound in response to an mRNA fragment containing the Kozak consensus sequence. The ribosomes adopt two globally different conformations that are related to each other by the rotation of the small subunit. Comparison with bacterial ribosome complexes reveals that the global structures and modes of intersubunit rotation of the yeast ribosome differ significantly from those in the bacterial counterpart, most notably in the regions involving the tRNA, small ribosomal subunit, and conserved helix 69 of the large ribosomal subunit. The structures provide insight into ribosome dynamics implicated in tRNA translocation and help elucidate the role of the Kozak fragment in positioning an open reading frame during translation initiation in eukaryotes

A Large Hadron Electron Collider at CERN

This document provides a brief overview of the recently published report on the design of the Large Hadron Electron Collider (LHeC), which comprises its physics programme, accelerator physics, technology and main detector concepts. The LHeC exploits and develops challenging, though principally existing, accelerator and detector technologies. This summary is complemented by brief illustrations of some of the highlights of the physics programme, which relies on a vastly extended kinematic range, luminosity and unprecedented precision in deep inelastic scattering. Illustrations are provided regarding high precision QCD, new physics (Higgs, SUSY) and electron-ion physics. The LHeC is designed to run synchronously with the LHC in the twenties and to achieve an integrated luminosity of O(100) fb$^{-1}$. It will become the cleanest high resolution microscope of mankind and will substantially extend as well as complement the investigation of the physics of the TeV energy scale, which has been enabled by the LHC

Клинико-генетические характеристики спинальной мышечной атрофии с преимущественным поражением ног, обусловленной мутациями в гене DYNC1H1

Background. Spinal muscle atrophies (SMA) are a group of diverse heterogenous diseases caused by mutations in several dozens of genes. A rare form of autosomal dominant SMA predominantly affects muscles of the lower extremities.The study objective is to describe clinical and genetic characteristics of Russia-living patients with SMA predominantly affecting muscles of the lower extremities caused by the DYNC1H1 gene mutation discovered by next-generation exome sequencing.Materials and methods. To diagnose the syndrome a complex of examination techniques was used: genealogical analysis, neurological examination, electromyography, and DNA diagnostics. Changes in the nucleotide sequence in the probands and their parents identified with massive parallel sequencing were studied using direct automatic sequencing with oligonucleotide primers.Results. Five (5) patients from 4 families with heterozygous mutations in the DYNC1H1 gene were identified. In the patients, one type of SMA predominantly affecting the lower extremities was assumed. Prior to exome sequencing, all patients were monitored for myelodysplasia diagnosis, and magnetic resonance imaging of the spine has showed protrusions and/or spondylolisthesis of the lumbar spine in 4 of the patients. The obtained results can demonstrate both hyperdiagnosis and that spinal pathology is one of the characteristic symptoms of SMA predominantly affecting the lower extremities.Conclusion. The obtained results allow to make an assumption about a wide range of clinical polymorphisms in patients with mutations of the DYNC1H1 gene. Apart from typical clinical manifestations of SMA predominantly affecting the lower extremities, patients can be diagnosed with hereditary motor and sensory neuropathy 2, myelodysplasia, and congenital arthrogryposis which has to be taken into account during diagnostic search.Введение. Спинальные мышечные атрофии (СМА) – группа генетически гетерогенных заболеваний, обусловленных мутациями в нескольких десятках генов. Одна из редких форм аутосомно-доминантных СМА характеризуется преимущественным поражением мышц нижних конечностей.Цель исследования – описание клинико-генетических характеристик больных, проживающих на территории России, со СМА с преимущественным поражением мышц нижних конечностей, обусловленной мутациями в гене DYNC1H1, выявленными в результате проведения секвенирования экзома нового поколения.Материалы и методы. Для диагностики синдрома использовали комплекс методов обследования: генеалогический анализ, неврологический осмотр, электронейромиографию и ДНК-диагностику. Выявленные при проведении массового параллельного секвенирования изменения нуклеотидной последовательности у пробандов и их родителей были исследованы методом прямого автоматического секвенирования с использованием олигонуклеотидных праймеров.Результаты. Выявлены 5 больных из 4 семей с гетерозиготными мутациями в гене DYNC1H1. У пациентов предполагался один из вариантов СМА с преимущественным поражением нижних конечностей. До проведения секвенирования экзома все больные наблюдались с диагнозом миелодисплазии, причем при проведении магнитно-резонансной томографии спинного мозга у 4 из них выявлялись протрузии и/или спондилолистез поясничных позвонков. Полученные результаты могут свидетельствовать как о гипердиагностике, так и о том, что патология позвоночника может быть одним из характерных симптомов СМА с преимущественным поражением мышц нижних конечностей.Заключение. Полученные результаты позволяют высказать предположение о значительном размахе клинического полиморфизма у больных с мутациями в гене DYNC1H1. Помимо типичных клинических проявлений СМА с преимущественным поражением мышц нижних конечностей, у пациентов могут диагностироваться наследственная моторно-сенсорная нейропатия 2-го типа, миелодисплазия и врожденный артрогрипоз, что необходимо учитывать при проведении диагностического поиска

Functional expression of SCN5A mutation R1628Q associated with Brugada syndrome

目的 体外构建含R1628Q突变的人心脏钠离子通道α亚基真核表达载体pHL-hH1-R1628Q。研究R1628Q突变对钠通道电生理性质的影响，阐明Brugada综合征的发病机制，为疾病的基因诊断和分子治疗提供新的思路。 方法 设计带突变位点的引物，以含有人心脏钠离子通道cDNA的质粒为模板经重叠延伸PCR法体外定点诱变，构建R1628Q突变型人心脏钠离子通道真核表达载体。经酶切和DNA测序验证后与心脏钠离子通道β1亚基质粒pEGFP-N3-SCN1B瞬时共同转染到HEK293，48小时后全细胞记录钠通道的电生理特性。 结果 1.DNA测序证实突变质粒pHL-hH1-R1628Q构建...Objective To explore the functional effect of R1628Q mutation on SCN5A,we construct human cardiac sodium channel alpha subunit cDNA(hH1) mutated recombinant pHL-hH1-R1628Q via site-directed mutagenesis in vitro, and study the current and electrophysiological properties of the sodium channel using patch-clamp whole-cell recording in HEK293 cell into which the hH1-WT or hH1-R1628Q is transfected. T...学位：医学硕士院系专业：医学院_内科学学号：2452010115328

CLIC Polarized Positron Source Based on Laser Compton Scattering

We describe a possible layout and parameters of a polarized positron source for CLIC, where the positrons are produced from polarized gamma rays created by Compton scattering of a 1.3-GeV electron beam off a YAG laser. This scheme is very energy effective using high finesse laser cavities in conjunction with an electron storage ring. We point out the differences with respect to a similar system proposed for the ILC

Codon Size Reduction as the Origin of the Triplet Genetic Code

The genetic code appears to be optimized in its robustness to missense errors and frameshift errors. In addition, the genetic code is near-optimal in terms of its ability to carry information in addition to the sequences of encoded proteins. As evolution has no foresight, optimality of the modern genetic code suggests that it evolved from less optimal code variants. The length of codons in the genetic code is also optimal, as three is the minimal nucleotide combination that can encode the twenty standard amino acids. The apparent impossibility of transitions between codon sizes in a discontinuous manner during evolution has resulted in an unbending view that the genetic code was always triplet. Yet, recent experimental evidence on quadruplet decoding, as well as the discovery of organisms with ambiguous and dual decoding, suggest that the possibility of the evolution of triplet decoding from living systems with non-triplet decoding merits reconsideration and further exploration. To explore this possibility we designed a mathematical model of the evolution of primitive digital coding systems which can decode nucleotide sequences into protein sequences. These coding systems can evolve their nucleotide sequences via genetic events of Darwinian evolution, such as point-mutations. The replication rates of such coding systems depend on the accuracy of the generated protein sequences. Computer simulations based on our model show that decoding systems with codons of length greater than three spontaneously evolve into predominantly triplet decoding systems. Our findings suggest a plausible scenario for the evolution of the triplet genetic code in a continuous manner. This scenario suggests an explanation of how protein synthesis could be accomplished by means of long RNA-RNA interactions prior to the emergence of the complex decoding machinery, such as the ribosome, that is required for stabilization and discrimination of otherwise weak triplet codon-anticodon interactions

Structural and functional basis for RNA cleavage by Ire1

BACKGROUND: The unfolded protein response (UPR) controls the protein folding capacity of the endoplasmic reticulum (ER). Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase) domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. RESULTS: This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing \u3e/=7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL) of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. CONCLUSIONS: Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L

Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1

<p>Abstract</p> <p>Background</p> <p>Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity.</p> <p>Results</p> <p>Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn²⁺and Cd²⁺, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors.</p> <p>Conclusions</p> <p>Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer.</p