Isolation, crystallization, and investigation of ribosomal protein S8 complexed with specific fragments of rRNA of bacterial or archaeal origin. Biochemistry 66

Study of the nature of protein-rRNA complexes is a topical problem of modern molecular biology. Structural studies of rRNA-protein complexes are the most direct and precise method of analysis of these interactions. Because ribosomal proteins are most conservative during evolution, their complexes with specific RNA fragments provide an interesting model for studying RNA-protein interactions. Ribosomal protein S8 from E. coli plays a key role in assembling the small ribosomal subunit The major region of protein S8 binding on 16S rRNA was determined by partial hydrolysis with restric tion endonucleases The binding sites of protein S8 on 16S rRNA are similar in E. coli and T. thermophilus. It was shown that ACCELERATED PUBLICATION 0006 2979/01/6609 0948$25.00 ©2001 MAIK "Nauka / Interperiodica" * To whom correspondence should be addressed. Vol. 66, No. 9, 2001, pp. 948 953. Translated from Biokhimiya, Vol. 66, No. 9, 2001, pp. 1165 1171. Original Russian Text Copyright © 2001 Abstract-The core ribosomal protein S8 binds to the central domain of 16S rRNA independently of other ribosomal proteins and is required for assembling the 30S subunit. It has been shown with E. coli ribosomes that a short rRNA fragment restrict ed by nucleotides 588 602 and 636 651 is sufficient for strong and specific protein S8 binding. In this work, we studied the complexes formed by ribosomal protein S8 from Thermus thermophilus and Methanococcus jannaschii with short rRNA frag ments isolated from the same organisms. The dissociation constants of the complexes of protein S8 with rRNA fragments were determined. Based on the results of binding experiments, rRNA fragments of different length were designed and syn thesized in preparative amounts in vitro using T7 RNA polymerase. Stable S8-RNA complexes were crystallized. Crystals were obtained both for homologous bacterial and archaeal complexes and for hybrid complexes of archaeal protein with bac terial rRNA. Crystals of the complex of protein S8 from M. jannaschii with the 37 nucleotide rRNA fragment from the same organism suitable for X ray analysis were obtained

Lagrange-Fedosov Nonholonomic Manifolds

We outline an unified approach to geometrization of Lagrange mechanics, Finsler geometry and geometric methods of constructing exact solutions with generic off-diagonal terms and nonholonomic variables in gravity theories. Such geometries with induced almost symplectic structure are modelled on nonholonomic manifolds provided with nonintegrable distributions defining nonlinear connections. We introduce the concept of Lagrange-Fedosov spaces and Fedosov nonholonomic manifolds provided with almost symplectic connection adapted to the nonlinear connection structure. We investigate the main properties of generalized Fedosov nonholonomic manifolds and analyze exact solutions defining almost symplectic Einstein spaces.Comment: latex2e, v3, published variant, with new S.V. affiliatio

Observation of enhanced chiral asymmetries in the inner-shell photoionization of uniaxially oriented methyloxirane enantiomers

Most large molecules are chiral in their structure: they exist as two enantiomers, which are mirror images of each other. Whereas the rovibronic sublevels of two enantiomers are almost identical, it turns out that the photoelectric effect is sensitive to the absolute configuration of the ionized enantiomer - an effect termed Photoelectron Circular Dichroism (PECD). Our comprehensive study demonstrates that the origin of PECD can be found in the molecular frame electron emission pattern connecting PECD to other fundamental photophysical effects as the circular dichroism in angular distributions (CDAD). Accordingly, orienting a chiral molecule in space enhances the PECD by a factor of about 10

Role of Polypyrimidine Tract Binding Protein in Mediating Internal Initiation of Translation of Interferon Regulatory Factor 2 RNA

BACKGROUND: Earlier we have reported translational control of interferon regulatory factor 2 (IRF2) by internal initiation (Dhar et al, Nucleic Acids Res, 2007). The results implied possible role of IRF2 in controlling the intricate balance of cellular gene expression under stress conditions in general. Here we have investigated the secondary structure of the Internal Ribosome Entry Site of IRF2 RNA and demonstrated the role of PTB protein in ribosome assembly to facilitate internal initiation. METHODOLOGY/PRINCIPAL FINDINGS: We have probed the putative secondary structure of the IRF2 5'UTR RNA using various enzymatic and chemical modification agents to constrain the secondary structure predicted from RNA folding algorithm Mfold. The IRES activity was found to be influenced by the interaction of trans-acting factor, polypyrimidine tract binding protein (PTB). Deletion of 25 nts from the 3'terminus of the 5'untranslated region resulted in reduced binding with PTB protein and also showed significant decrease in IRES activity compared to the wild type. We have also demonstrated putative contact points of PTB on the IRF2-5'UTR using primer extension inhibition assay. Majority of the PTB toe-prints were found to be restricted to the 3'end of the IRES. Additionally, Circular Dichroism (CD) spectra analysis suggested change in the conformation of the RNA upon PTB binding. Further, binding studies using S10 extract from HeLa cells, partially silenced for PTB gene expression, resulted in reduced binding by other trans-acting factors. Finally, we have demonstrated that addition of recombinant PTB enhances ribosome assembly on IRF2 IRES suggesting possible role of PTB in mediating internal initiation of translation of IRF2 RNA. CONCLUSION/SIGNIFICANCE: It appears that PTB binding to multiple sites within IRF2 5'UTR leads to a conformational change in the RNA that facilitate binding of other trans-acting factors to mediate internal initiation of translation

Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius

Central to genetic work in any organism are the availability of a range of inducible and constitutive promoters. In this work we studied several promoters for use in the hyperthermophilic archaeon Sulfolobus acidocaldarius. The promoters were tested with the aid of an E. coli–Sulfolobus shuttle vector in reporter gene experiments. As the most suitable inducible promoter a maltose inducible promoter was identified. It comprises 266 bp of the sequence upstream of the gene coding for the maltose/maltotriose binding protein (mbp, Saci_1165). Induction is feasible with either maltose or dextrin at concentrations of 0.2–0.4%. The highest increase in expression (up to 17-fold) was observed in late exponential and stationary phase around 30–50 h after addition of dextrin. Whereas in the presence of glucose and xylose higher basal activity and reduced inducibility with maltose is observed, sucrose can be used in the growth medium additionally without affecting the basal activity or the inducibility. The minimal promoter region necessary could be narrowed down to 169 bp of the upstream sequence. The ABCE1 protein from S. solfataricus was successfully expressed under control of the inducible promoter with the shuttle vector pC and purified from the S. acidocaldarius culture with a yield of about 1 mg L−1 culture. In addition we also determined the promoter strength of several constitutive promoters

Unfolding the Neutron Flux Spectrum on the Surface of Mars Using the MSL‐RAD and Odyssey‐HEND Data

Understanding the long-term radiation environment at the surface of Mars allows us to estimate the exposure for future robotic and crewed missions. Typically, the radiation environment includes charged particles (i.e., protons and heavier ions) and neutral particles (i.e., gamma rays and secondary neutrons). Previous studies used in-situ measurements, models, or both to determine the characteristics of the radiation at Mars. For example, the Mars Science Laboratory instrument, the Radiation Assessment Detector (RAD), has provided invaluable in-situ data since landing in 2012. However, the RAD instrument is only sensitive to neutrons with energies > ∼6 MeV and therefore misses what is expected to be a substantial flux of lower-energy neutrons. To address this gap, we have developed an approach to derive the surface neutron spectrum using the MSL RAD data augmented by orbital data from the High Energy Neutron Detector (HEND) onboard Mars Odyssey (neutron energy < ∼10 MeV). Using a power law fit, we determine neutron flux spectra that reproduce the measurements recorded by both RAD and HEND. Our approach involves a series of Monte Carlo simulations to develop a set of atmospheric transmission functions that enables us to convert the on-orbit HEND data to their corresponding surface neutron flux spectra. The combined RAD—HEND data present a unique opportunity to obtain a complete picture of the surface neutron environment

Optical and Spin Properties of NV Center Ensembles in Diamond Nano Pillars

Nitrogen vacancy NV color centers in diamond are excellent quantum sensors possessing high sensitivity and nano scale spatial resolution. Their integration in photonic structures is often desired, since it leads to an increased photon emission and also allows the realization of solid state quantum technology architectures. Here, we report the fabrication of diamond nano pillars with diameters up to 1000 nm by electron beam lithography and inductively coupled plasma reactive ion etching in nitrogen rich diamonds type Ib with [100] and [111] crystal orientations. The NV centers were created by keV He ion bombardment and subsequent annealing, and we estimate an average number of NVs per pillar to be 4300 300 and 520 120 for the [100] and [111] samples, respectively. Lifetime measurements of the NVs excited state showed two time constants with average values of amp; 964;1 amp; 8776; 2 ns and amp; 964;2 amp; 8776; 8 ns, which are shorter as compared to a single color center in a bulk crystal amp; 964; amp; 8776; 10 ns . This is probably due to a coupling between the NVs as well as due to interaction with bombardment induced defects and substitutional nitrogen P1 centers . Optically detected magnetic resonance measurements revealed a contrast of about 5 and average coherence and relaxation times of T2 [100] 420 40 ns, T2 [111] 560 50 ns, and T1 [100] 162 11 amp; 956;s, T1 [111] 174 24 amp; 956;s. These pillars could find an application for scanning probe magnetic field imagin

Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol

Chemical mapping methods probe RNA structure by revealing and leveraging correlations of a nucleotide's structural accessibility or flexibility with its reactivity to various chemical probes. Pioneering work by Lucks and colleagues has expanded this method to probe hundreds of molecules at once on an Illumina sequencing platform, obviating the use of slab gels or capillary electrophoresis on one molecule at a time. Here, we describe optimizations to this method from our lab, resulting in the MAP-seq protocol (Multiplexed Accessibility Probing read out through sequencing), version 1.0. The protocol permits the quantitative probing of thousands of RNAs at once, by several chemical modification reagents, on the time scale of a day using a table-top Illumina machine. This method and a software package MAPseeker (http://simtk.org/home/map_seeker) address several potential sources of bias, by eliminating PCR steps, improving ligation efficiencies of ssDNA adapters, and avoiding problematic heuristics in prior algorithms. We hope that the step-by-step description of MAP-seq 1.0 will help other RNA mapping laboratories to transition from electrophoretic to next-generation sequencing methods and to further reduce the turnaround time and any remaining biases of the protocol.Comment: 22 pages, 5 figure

From music to mathematics and backwards: introducing algebra, topology and category theory into computational musicology

International audienceDespite a long historical relationship between mathematics and music, the interest of mathematicians is a recent phenomenon. In contrast to statistical methods and signal-based approaches currently employed in MIR (Music Information Research), the research project described in this paper stresses the necessity of introducing a structural multidisciplinary approach into computational musicology making use of advanced mathematics. It is based on the interplay between three main mathematical disciplines: algebra, topology and category theory. It therefore opens promising perspectives on important prevailing challenges, such as the automatic classification of musical styles or the solution of open mathematical conjectures, asking for new collaborations between mathematicians, computer scientists, musicologists, and composers. Music can in fact occupy a strategic place in the development of mathematics since music-theoretical constructions can be used to solve open mathematical problems. The SMIR project also differs from traditional applications of mathematics to music in aiming to build bridges between different musical genres, ranging from contemporary art music to popular music, including rock, pop, jazz and chanson. Beyond its academic ambition, the project carries an important societal dimension stressing the cultural component of 'mathemusical' research, that naturally resonates with the underlying philosophy of the “Imagine Maths”conference series. The article describes for a general public some of the most promising interdisciplinary research lines of this project

Enantiosensitive Structure Determination by Photoelectron Scattering on Single Molecules

X-ray as well as electron diffraction are powerful tools for structure determination of molecules. Electron diffraction methods yield \r{A}ngstrom-resolution even when applied to large systems or systems involving weak scatterers such as hydrogen atoms. For cases in which molecular crystals cannot be obtained or the interaction-free molecular structure is to be addressed, corresponding electron scattering approaches on gas-phase molecules exist. Such studies on randomly oriented molecules, however, can only provide information on interatomic distances, which is challenging to analyse in case of overlapping distance parameters and they do not reveal the handedness of chiral systems8. Here, we present a novel scheme to obtain information on the structure, handedness and even detailed geometrical features of single molecules in the gas phase. Using a loop-like analysis scheme employing input from ab initio computations on the photoionization process, we are able to deduce the three dimensional molecular structure with sensitivity to the position individual atoms, as e.g. protons. To achieve this, we measure the molecular frame diffraction pattern of core-shell photoelectrons in combination with only two ionic fragments from a molecular Coulomb explosion. Our approach is expected to be suitable for larger molecules, as well, since typical size limitations regarding the structure determination by pure Coulomb explosion imaging are overcome by measuring in addition the photoelectron in coincidence with the ions. As the photoelectron interference pattern captures the molecular structure at the instant of ionization, we anticipate our approach to allow for tracking changes in the molecular structure on a femtosecond time scale by applying a pump-probe scheme in the future