eIF4G stimulates the activity of the DEAD box protein eIF4A by a conformational guidance mechanism

The activity of eIF4A, a key player in translation initiation, is regulated by other translation factors through currently unknown mechanisms. Here, we provide the necessary framework to understand the mechanism of eIF4A's regulation by eIF4G. In solution, eIF4A adopts a defined conformation that is different from the crystal structure. Binding of eIF4G induces a ‘half-open' conformation by interactions with both domains, such that the helicase motifs are pre-aligned for activation. A primary interface acts as an anchor for complex formation. We show here that formation of the secondary interface is essential for imposing the ‘half-open' conformation on eIF4A, and it is critical for the functional interaction of eIF4G with eIF4A. Via this bipartite interaction, eIF4G guides the transition of eIF4A between the ‘half-open' and closed conformations, and stimulates its activity by accelerating the rate-limiting step of phosphate release. Subtle changes induced by eIF4G may be amplified by input signals from other translation factors, leading to an efficient regulation of translation initiatio

eIF4G stimulates the activity of the DEAD box protein eIF4A by a conformational guidance mechanism

The activity of eIF4A, a key player in translation initiation, is regulated by other translation factors through currently unknown mechanisms. Here, we provide the necessary framework to understand the mechanism of eIF4A’s regulation by eIF4G. In solution, eIF4A adopts a defined conformation that is different from the crystal structure. Binding of eIF4G induces a ‘half-open’ conformation by interactions with both domains, such that the helicase motifs are pre-aligned for activation. A primary interface acts as an anchor for complex formation. We show here that formation of the secondary interface is essential for imposing the ‘half-open’ conformation on eIF4A, and it is critical for the functional interaction of eIF4G with eIF4A. Via this bipartite interaction, eIF4G guides the transition of eIF4A between the ‘half-open’ and closed conformations, and stimulates its activity by accelerating the rate-limiting step of phosphate release. Subtle changes induced by eIF4G may be amplified by input signals from other translation factors, leading to an efficient regulation of translation initiation

Light-induced absorption changes in ferroelectric crystals:SrxBa1-xNb2O6:Ce; KTaO3; KTa1-xNbxO3

The aim of the current work was to investigate the photo-induced charge transport at low temperatures, allowing more sensitive, detailed measurements of the first steps in the build-up of space charge fields, which modify the refractive index, leading to modern applications like volume holographic storage. We investigated the light-induced properties of SBN:Ce, KTO and KTN materials like origin of trapping centers which are involved in the charge transport process, characterization of trapping centers, like temperature dependence, illumination intensity dependence, evolution with time, spectral response, activation energies, the basic properties of the electronic excitations and photo-carriers localization based on results of absorption, light-induced absorption, photoluminescence, and photocurrent. The main contributions of this dissertation are summarized as follows: The experimental intensity dependence, temperature dependence, and decay process of the light-induced polaron (NIR) and VIS center absorption can be fitted with the help of a simplified charge transfer model (for SBN). The decay observed of the NIR polaron and the VIS centers is present due to the Fourier spectrometer light. The dissociation of the VIS centers into NIR centers under red light was observed. The model proposed for the VIS-centers in SBN is a triad structure related to the simultaneous bonding of two hole polarons and one electronic polaron.In KTN the emergence of the UV-light induced wide absorption bands in the NIR region with maxima at 0.69 0.8 eV at low temperatures is treated as a manifestation of the localization of photo-induced electrons and the formation of small electron polarons in close-neighbor Nb-Nb pair centers. Also, these properties in KTN can be fitted with the help of the simplified charge transfer model

DNA-induced narrowing of the gyrase N-gate coordinates T-segment capture and strand passage

DNA gyrase introduces negative supercoils into DNA in an ATP-dependent reaction. DNA supercoiling is catalyzed by a strand-passage mechanism, in which a T-segment of DNA is passed through the gap in a transiently cleaved G-segment. Strand passage requires the coordinated closing and opening of three protein interfaces in gyrase, the N-gate, DNA-gate, and C-gate. We show here that DNA binding to the DNA-gate of gyrase and wrapping of DNA around the C-terminal domains of GyrA induces a narrowing of the N-gate. This half-closed state prepares capture of a T-segment in the upper cavity of gyrase. Subsequent N-gate closure upon binding of ATP then poises the reaction toward strand passage. The N-gate reopens after ATP hydrolysis, allowing for further catalytic cycles. DNA binding, cleavage, and wrapping and N-gate narrowing are intimately linked events that coordinate conformational changes at the DNA and the N-gate

The DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

Gyrase is the only type II topoisomerase that introduces negative supercoils into DNA. Supercoiling is catalyzed via a strand-passage mechanism, in which the gate DNA (gDNA) is transiently cleaved, and a second DNA segment, the transfer DNA (tDNA), is passed through the gap before the gDNA is religated. Strand passage requires an opening of the so-called DNA-gate by ≈2 nm. A single-molecule FRET study reported equal populations of open and closed DNA-gate in topoisomerase II. We present here single-molecule FRET experiments that monitor the conformation of DNA bound to the DNA-gate of Bacillus subtilis gyrase and the conformation of the DNA-gate itself. DNA bound to gyrase adopts two different conformations, one slightly, one severely distorted. DNA distortion requires cleavage, but neither ATP nor the presence of a tDNA. At the same time, the DNA-gate of gyrase is predominantly in the closed conformation. In agreement with the single molecule data and with the danger of dsDNA breaks for genome integrity, <5% of cleavage complexes are detected in equilibrium. Quinolone inhibitors favor DNA cleavage by B. subtilis gyrase, but disfavor DNA distortion, and the DNA-gate remains in the closed conformation. Our results demonstrate that DNA binding, distortion and cleavage, and gate-opening are mechanistically distinct events. During the relaxation and supercoiling reactions, gyrase with an open DNA-gate is not significantly populated, consistent with gate-opening as a very rare event that only occurs briefly to allow for strand passage