Structural Diversity in Bacterial Ribosomes: Mycobacterial 70S Ribosome Structure Reveals Novel Features

Here we present analysis of a 3D cryo-EM map of the 70S ribosome from Mycobacterium smegmatis, a saprophytic cousin of the etiological agent of tuberculosis in humans, Mycobacterium tuberculosis. In comparison with the 3D structures of other prokaryotic ribosomes, the density map of the M. smegmatis 70S ribosome reveals unique structural features and their relative orientations in the ribosome. Dramatic changes in the periphery due to additional rRNA segments and extra domains of some of the peripheral ribosomal proteins like S3, S5, S16, L17, L25, are evident. One of the most notable features appears in the large subunit near L1 stalk as a long helical structure next to helix 54 of the 23S rRNA. The sharp upper end of this structure is located in the vicinity of the mRNA exit channel. Although the M. smegmatis 70S ribosome possesses conserved core structure of bacterial ribosome, the new structural features, unveiled in this study, demonstrates diversity in the 3D architecture of bacterial ribosomes. We postulate that the prominent helical structure related to the 23S rRNA actively participates in the mechanisms of translation in mycobacteria

The Cryo-EM Structure of a Complete 30S Translation Initiation Complex from Escherichia coli

Formation of the 30S initiation complex (30S IC) is an important checkpoint in regulation of gene expression. The selection of mRNA, correct start codon, and the initiator fMet-tRNAfMet requires the presence of three initiation factors (IF1, IF2, IF3) of which IF3 and IF1 control the fidelity of the process, while IF2 recruits fMet-tRNAfMet. Here we present a cryo-EM reconstruction of the complete 30S IC, containing mRNA, fMet-tRNAfMet, IF1, IF2, and IF3. In the 30S IC, IF2 contacts IF1, the 30S subunit shoulder, and the CCA end of fMet-tRNAfMet, which occupies a novel P/I position (P/I1). The N-terminal domain of IF3 contacts the tRNA, whereas the C-terminal domain is bound to the platform of the 30S subunit. Binding of initiation factors and fMet-tRNAfMet induces a rotation of the head relative to the body of the 30S subunit, which is likely to prevail through 50S subunit joining until GTP hydrolysis and dissociation of IF2 take place. The structure provides insights into the mechanism of mRNA selection during translation initiation

Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type—WT) occur at 4–7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures

Терапевтическая эффективность внутриартериального введения нейральных прогениторных клеток, полученных из индуцированных плюрипотентных стволовых клеток, при остром экспериментальном ишемическом инсульте у крыс

Aim. Neural progenitor cells (NPC) are used for the development of cell therapies of neurological diseases. Their stereotaxic transplantation in the middle cerebral artery occlusion (MCAO) model imitating ischemic stroke results in symptom aleviation. However, exploration of less invasive transplantation options is essential, because stereotaxic transplantation is a complex procedure and can be applied to humans only by vital indications in a specialized neurological ward. The aim of the present study was to evaluate the efficacy of cell therapy of the experimental ischemic stroke by the intra-arterial transplantation of NPC.Materials and methods. NPC for transplantation (IPSC-NPC) were derived by two-stage differentiation of cells of a stable line of human induced pluripotent stem cells. Stroke modeling in rats was carried out by transitory 90 min endovascular MCAO by a silicon-tipped filament. NPC were transplanted 24 hours after MCAO. Repetitive magnetic resonance tomography of experimental animals was made with the Bruker BioSpin ClinScan tomograph with 7 Tl magnetic field induction. Animal survival rate and neurological deficit (using mNSS standard stroke severity scale) were evaluated at the 1st (before IPSC-NPC transplantation), 7th and 14th day after transplantation. Histological studies were carried out following standard protocols.Results. Intra-arterial transplantation of 7 × 105 IPSC-NPC in 1 ml at a constant 100 l/min rate in case of secured blood flow through the internal carotid artery did not cause brain capillary embolism, additional cytotoxic brain tissue edemas or other complications, while inducing increase of animal survival rate and enhanced revert of the neurological deficit. IPSC-NPC accumulation in brain after intra-arterial infusion was demonstrated. Some cells interacted with the capillary endothelium and probably penetrated through the blood-brain barrier.Conclusion. Therapeutic efficacy of the systemic, intra-arterial administration of NPC in ischemic stroke has been experimentally proven. A method of secure intra-arterial infusion of cell material into the internal carotid artery middle in rats has been developed and tested.Цель. Нейральные прогениторные клетки (НПК) используются при разработке технологий клеточной терапии неврологических заболеваний. Их стереотаксическое введение в мозг крыс после имитирующей ишемический инсульт операции окклюзии средней мозговой артерии (ОСМА) приводит к облегчению симптоматики. Однако стереотаксическое введение является сложной процедурой и для лечения болезней человека может быть применено только в специализированной клинике по жизненным показаниям, что делает необходимым исследование возможности менее травматичных способов трансплантации. Цель настоящей работы – исследование возможности проведения клеточной терапии экспериментального инсульта путем внутриартериального введения НПК.Материалы и методы. НПК для трансплантации (ИПСК-НПК) получали путем двухступенчатой дифференцировки клеток стабильной линии индуцированных плюрипотентных стволовых клеток человека. Моделирование инсульта у крыс производилось методом транзиторной (90 мин) эндоваскулярной ОСМА филаментом с силиконовым наконечником. Внутриартериальная трансплантация НПК выполнялась через 24 часа после ОСМА. Магнитно-резонансная томография экспериментальных животных в динамике проводилась на МР-томографе ClinScan фирмы Bruker BioSpin с индукцией магнитного поля 7 Тл. На 1 (до введения ИПСК-НПК), 7 и 14-е сутки после трансплантации оценивались выживаемость животных и неврологический дефицит с использованием стандартной шкалы оценки тяжести инсульта mNSS для грызунов. Гистологические исследования проводили, пользуясь стандартными методами.Результаты. Внутриартериальная трансплантация ИПСК-НПК в дозе 7 × 105 НПК в 1 мл с равномерной скоростью100 мкл/мин и поддержанием кровотока по внутренней сонной артерии не вызывала эмболии капилляров мозга, появления новых зон цитотоксического отека вещества головного мозга или других осложнений и приводила к достоверному повышению выживаемости животных и более быстрому восстановлению неврологического статуса. Продемонстрировано накопление ИПСК-НПК в мозге после их внутриартериальной инфузии. Часть клеток взаимодействовала с эндотелием капилляров и, вероятно, способна проникать через ГЭБ.Заключение. Получено экспериментальное подтверждение терапевтической эффективности НПК при ишемическом инсульте при системной, внутриартериальной трансплантации. Отработан и протестирован метод безопасной внутриартериальной инфузии клеточного материала в бассейн внутренней сонной артерии у крыс

Major rearrangements in the 70S ribosomal 3D structure caused by a conformational switch in 16S ribosomal RNA.

Dynamic changes in secondary structure of the 16S rRNA during the decoding of mRNA are visualized by three-dimensional cryo-electron microscopy of the 70S ribosome. Thermodynamically unstable base pairing of the 912-910 (CUC) nucleotides of the 16S RNA with two adjacent complementary regions at nucleotides 885-887 (GGG) and 888-890 (GAG) was stabilized in either of the two states by point mutations at positions 912 (C912G) and 885 (G885U). A wave of rearrangements can be traced arising from the switch in the three base pairs and involving functionally important regions in both subunits of the ribosome. This significantly affects the topography of the A-site tRNA-binding region on the 30S subunit and thereby explains changes in tRNA affinity for the ribosome and fidelity of decoding mRNA

The Ribosome - Three-Dimensional Structure and Ligand-Binding Studies

To date, cryo-electron microscopy has become the most successful technique for exploring the structure of the ribosome and for studying binding positions of its various ligands, with the resolution slowly extending toward 10 Å. Obstacles in the attempts to improve resolution are the limited stability and coherence of the electron microscope, the statistics of data collection, and the conformational heterogeneity of the specimen. The last factor in this list proved to be the reason why it has been difficult to go past 18-20 Å with many specimens despite the use of state-of-the-art electron microscopes and inclusion of tens of thousand of projections. A breakthrough has been achieved with a protein synthesis initiation-like complex in which mRNA and fMet-tRNA is bound to the E. coli ribosome. The high occupancy and extraordinary conformational homogeneity of this specimen has enabled us to reach a resolution of 15 Å