Conformation transitions of eukaryotic polyribosomes during multi-round translation.

Using sedimentation and cryo electron tomography techniques, the conformations of eukaryotic polyribosomes formed in a long-term cell-free translation system were analyzed over all the active system lifetime (20-30 translation rounds during 6-8 h in wheat germ extract at 25°C). Three distinct types of the conformations were observed: (i) circular polyribosomes, varying from ring-shaped forms to circles collapsed into double rows, (ii) linear polyribosomes, tending to acquire planar zigzag-like forms and (iii) densely packed 3D helices. At the start, during the first two rounds of translation mostly the circular (ring-shaped and double-row) polyribosomes and the linear (free-shaped and zigzag-like) polyribosomes were formed ('juvenile phase'). The progressive loading of the polyribosomes with translating ribosomes induced the opening of the circular polyribosomes and the transformation of a major part of the linear polyribosomes into the dense 3D helices ('transitional phase'). After 2 h from the beginning (about 8-10 rounds of translation) this compact form of polyribosomes became predominant, whereas the circular and linear polyribosome fractions together contained less than half of polysomal ribosomes ('steady-state phase'). The latter proportions did not change for several hours. Functional tests showed a reduced translational activity in the fraction of the 3D helical polyribosomes.

Phenomenological thermodynamics and the structure formation mechanism of the CuTi2S4 rhombohedral phase.

The theory of structural phase transition in CuTi2S4 is proposed. The symmetry of order parameters, thermodynamics and the mechanism of the atomic structure formation of the rhombohedral Cu-Ti-thiospinel have been studied. The critical order parameter inducing the phase transition has been found. Within the Landau theory of phase transitions, it is shown that the phase state may change from the high-symmetry cubic disordered Fd3[combining macron]m phase to the low-symmetry ordered rhombohedral R3[combining macron]m phase as a result of phase transition of the first order close to the second order. It is shown that the rhombohedral structure of CuTi2S4 is formed as a result of the displacements of all types of atoms and the ordering of Cu-atoms (1 : 1 order type in tetrahedral spinel sites), Ti-atoms (1 : 1 : 6 order type in octahedral spinel sites), and S-atoms (1 : 1 : 3 : 3 order type). The Cu- and Ti-atoms form metal nanoclusters which are named a "bunch" of dimers. The "bunch" of dimers in CuTi2S4 is a new type of self-organization of atoms in frustrated spinel-like structures. It is shown that Ti-atoms also form other types of metal nanoclusters: trimers and tetrahedra.

Formation of circular polyribosomes on eukaryotic mRNA without cap-structure and poly(A)-tail: a cryo electron tomography study.

The polyribosomes newly formed on recombinant GFP-encoding mRNAs in a wheat germ cell-free translation system were analyzed using cryo-electron tomography, with sub-tomogram averaging of polysomal ribosomes and reconstruction of 3D structures of individual polyribosomes. The achieved level of resolution in the reconstructed polyribosomes allowed deducing the mRNA path by connecting adjacent exit and entry sites at the ribosomes inside each polyribosome. In this way, the circularity of a significant fraction (about 50%) of translating polyribosomes was proved in the case of the capped poly(A)-tailed mRNA, in agreement with the existing paradigm of the circularization via interaction of cap-bound initiation factor eIF4F with poly(A)-binding protein. However, translation of the capped mRNA construct without poly(A) tail, but with unspecific 3'-UTR derived from non-coding plasmid sequence, also led to the formation of circular polyribosomes in similar proportion (40%). Moreover, the polyribosomes formed on the uncapped non-polyadenylated mRNA with non-synergistic 5'- and 3'-UTRs proved to be circular as well, and appeared in the same proportion as in the previous cases. Thus, the formation of circular polyribosomes was found to be virtually independent of the presence of cap structure and poly(A) tail in mRNA, in contrast to the longstanding paradigm in the field.

Efficiency of Generation of Surface Acoustic Waves in Barium Strontium Titanate Thin Films.

The excitation of surface acoustic waves (SAWs) on the surface of the ferroelectric film [barium strontium titanate (BST)] located on the dielectric substrate (silicon) was studied theoretically. We found that the most effective SAW excitation takes place when spontaneous polarization occurs in the film plane and the wave propagates along the direction adjacent to the direction of the spontaneous polarization vector. Based on a nonlinear model of phase transitions in solid BST solutions, the dependency of the material constants of piezo-effect equations on the misfit strain with a fixed concentration was obtained numerically. The effect of various misfit strains on SAW characteristics was studied for the film located on single-crystal silicon. It was shown that the effectiveness of SAW excitation increases as the misfit strain nears the boundary of phase transition.

230Th dating of flowstone from Ignatievskaya Cave, Russia: Age constraints of rock art and paleoclimate inferences.

Paleolithic antiquity of parietal art in Ignatievskaya cave, Southern Ural, is supported by its subject (Late Pleistocene animals) as well as by paleontological and palynological data, and 14C dates from cultural layers associated with artistic activity (17.8-16.3 cal ka BP; association is established by finds of ochre in these layers). However, three 14C dates of charcoal motifs yielded younger, Holocene ages (7.4-6.0 cal ka BP). In this study, we constrain the age of parietal art in the cave by 230Th dating of flowstone that brackets the paintings. Flowstone did not form in the cave between c. 78 and 10 ka BP, due to widespread permafrost in northern Eurasia at that time. Our 230Th dates do not support the middle Holocene age of art in Ignatievskaya cave and are consistent with its Upper Paleolithic antiquity instead.

Step-wise formation of eukaryotic double-row polyribosomes and circular translation of polysomal mRNA.

The time course of polysome formation was studied in a long-term wheat germ cell-free translation system using sedimentation and electron microscopy techniques. The polysomes were formed on uncapped luciferase mRNA with translation-enhancing 5' and 3' UTRs. The formation of fully loaded polysomes was found to be a long process that required many rounds of translation and proceeded via several phases. First, short linear polysomes containing no more than six ribosomes were formed. Next, folding of these polysomes into short double-row clusters occurred. Subsequent gradual elongation of the clusters gave rise to heavy-loaded double-row strings containing up to 30-40 ribosomes. The formation of the double-row polysomes was considered to be equivalent to circularization of polysomes, with antiparallel halves of the circle being laterally stuck together by ribosome interactions. A slow exchange with free ribosomes and free mRNA observed in the double-row type polysomes, as well as the resistance of translation in them to AMP-PNP, provided evidence that most polysomal ribosomes reinitiate translation within the circularized polysomes without scanning of 5' UTR, while de novo initiation including 5' UTR scanning proceeds at a much slower rate. Removal or replacements of 5' and 3' UTRs affected the initial phase of translation, but did not prevent the formation of the double-row polysomes during translation.

Tunable Electromechanical Properties of a Barium Strontium Titanate Ferroelectric Film Under a Uniaxial Stress.

The possibility of the development of MEMS devices based on the tunable ferroelectric film Ba 0.8Sr 0.2O3 properties under uniaxial deformation was studied theoretically. The thermodynamic model of the phase transitions for the film under uniaxial stress was constructed. The behavior of the material constants for the film in various phase states was investigated. The propagation properties of the surface acoustic wave (SAW) under the uniaxial stress were studied for the film located on the single-crystal silicon substrate. It was shown that the SAW resonance frequency changes within 3 MHz for the frequency of 274 MHz, and 9 MHz for the frequency of 512 MHz.

Phase transitions in Bi4Ti3O12.

Bi4Ti3O12 is a representative of the Aurivillius family of layered perovskites. These are high-temperature ferroelectric materials with prospects for applications in random-access memory and are characterized by an extremely confused interaction of their structural degrees of freedom. Using group-theoretical methods, structural distortions in the Bi4Ti3O12 high-symmetry phase, caused by rotations of rigid octahedra and their displacements as a single unit, have been investigated, taking into account the connections between them. Within the Landau theory, a stable thermodynamic model of phase transitions with three order parameters has been constructed. It is shown that, according to the phenomenological phase diagram, the transition between the high-temperature tetragonal phase and the low-temperature ferroelectric can occur both directly and through intermediate states, including those observed experimentally. The role of improper ordered parameters and possible domain configurations in the structure formation of the low-temperature ferroelectric phase are discussed.

Vanadium clusters formation in geometrically frustrated spinel oxide AlV2O4.

The spinel oxide AlV2O4 is a unique material, in which the formation of clusters is accompanied by atomic, charge and orbital ordering and a rhombohedral lattice distortion. In this work a theory of the structural phase transition in AlV2O4 is proposed. This theory is based on the study of the order-parameter symmetry, thermodynamics, electron density distribution, crystal chemistry and mechanisms of formation of the atomic and orbital structures of the rhombohedral phase. It is established that the critical order parameter is transformed according to irreducible representation k9(τ4) (in Kovalev notation) of the Fd \bar{3}m space group. Knowledge of the order-parameter symmetry allows us to show that the derived AlV2O4 rhombohedral structure is a result of displacements of all atom types and the ordering of Al atoms (1:1 order type in tetrahedral spinel sites), V atoms (1:1:6 order type in octahedral sites) and O atoms (1:1:3:3 order type), and the ordering of dxy, dxz and dyz orbitals. Application of the density functional theory showed that V atoms in the Kagomé sublattice formed separate trimers. Also, no sign of metallic bonding between separate vanadium trimers in the heptamer structure was found. The density functional theory study and the crystal chemical analysis of V-O bond lengths allowed us to assume the existence of dimers and trimers as main clusters in the structure of the AlV2O4 rhombohedral modification. The trimer model of the low-symmetry AlV2O4 structure is proposed. Within the Landau theory of phase transitions, typical diagrams of possible phase states are built. It is shown that phase states can be changed as a first-order phase transition close to the second order in the vicinity of tricritical points of the phase diagrams.

Continuous-exchange protein-synthesizing systems.

Protein synthesis in cell-free systems is an emerging technology already competing with in vivo expression methods. In this chapter the basic principles of continuous-exchange protein synthesizing systems, and protocols for Escherichia coli and wheat germ translation and transcription-translation systems are described. The ways to improve substrate supply in cell-free systems and mRNA design for eukaryotic system are discussed. Correct folding of the synthesized protein is demonstrated and discussed in detail.

The molecular structure of the left-handed supra-molecular helix of eukaryotic polyribosomes.

During protein synthesis, several ribosomes bind to a single messenger RNA (mRNA) forming large macromolecular assemblies called polyribosomes. Here we report the detailed molecular structure of a 100 MDa eukaryotic poly-ribosome complex derived from cryo electron tomography, sub-tomogram averaging and pseudo-atomic modelling by crystal structure fitting. The structure allowed the visualization of the three functional parts of the polysome assembly, the central core region that forms a rather compact left-handed supra-molecular helix, and the more open regions that harbour the initiation and termination sites at either ends. The helical region forms a continuous mRNA channel where the mRNA strand bridges neighbouring exit and entry sites of the ribosomes and prevents mRNA looping between ribosomes. This structure provides unprecedented insights into protein- and RNA-mediated inter-ribosome contacts that involve conserved sites through 40S subunits and long protruding RNA expansion segments, suggesting a role in stabilizing the overall polyribosomal assembly.

Membrane protein expression in cell-free systems.

Cell-free expression has emerged as a promising tool for the fast and efficient production of membrane proteins. The rapidly growing number of successfully produced targets in combination with the continuous development of new applications significantly promotes the distribution of this technology. Membrane protein synthesis by cell-free expression does not appear to be restricted by origin, size or topology of the target, and its global application is therefore a highly valuable characteristic. The technology is relatively fast to establish in standard biochemical labs, and it does not require expensive equipment. Moreover, it enables the production of membrane proteins in completely new modes, like the direct translation into detergent micelles, which is not possible with any other expression system. In this protocol, we focus on the currently most efficient cell-free expression system for membrane proteins based on Escherichia coli extracts.

Strategies for the cell-free expression of membrane proteins.

Cell-free expression offers an interesting alternative method to produce membrane proteins in high amounts. Elimination of toxicity problems, reduced proteolytic degradation and a nearly unrestricted option to supply potentially beneficial compounds like cofactors, ligands or chaperones into the reaction are general advantages of cell-free expression systems. Furthermore, the membrane proteins may be translated directly into appropriate hydrophobic and membrane-mimetic surrogates, which might offer significant benefits for the functional folding of the synthesized proteins. Cell-free expression is a rapidly developing and highly versatile technique and several systems of both, prokaryotic and eukaryotic origins, have been established. We provide protocols for the cell-free expression of membrane proteins in different modes including their expression as precipitate as well as their direct synthesis into detergent micelles or lipid bilayers.