TbMP42 is a structure-sensitive ribonuclease that likely follows a metal ion catalysis mechanism

RNA editing in African trypanosomes is characterized by a uridylate-specific insertion and/or deletion reaction that generates functional mitochondrial transcripts. The process is catalyzed by a multi-enzyme complex, the editosome, which consists of approximately 20 proteins. While for some of the polypeptides a contribution to the editing reaction can be deduced from their domain structure, the involvement of other proteins remains elusive. TbMP42, is a component of the editosome that is characterized by two C2H2-type zinc-finger domains and a putative oligosaccharide/oligonucleotide-binding fold. Recombinant TbMP42 has been shown to possess endo/exoribonuclease activity in vitro; however, the protein lacks canonical nuclease motifs. Using a set of synthetic gRNA/pre-mRNA substrate RNAs, we demonstrate that TbMP42 acts as a topology-dependent ribonuclease that is sensitive to base stacking. We further show that the chelation of Zn2+ cations is inhibitory to the enzyme activity and that the chemical modification of amino acids known to coordinate Zn2+ inactivates rTbMP42. Together, the data are suggestive of a Zn2+-dependent metal ion catalysis mechanism for the ribonucleolytic activity of rTbMP42

Predicting RNA pseudoknot folding thermodynamics

Based on the experimentally determined atomic coordinates for RNA helices and the self-avoiding walks of the P (phosphate) and C(4) (carbon) atoms in the diamond lattice for the polynucleotide loop conformations, we derive a set of conformational entropy parameters for RNA pseudoknots. Based on the entropy parameters, we develop a folding thermodynamics model that enables us to compute the sequence-specific RNA pseudoknot folding free energy landscape and thermodynamics. The model is validated through extensive experimental tests both for the native structures and for the folding thermodynamics. The model predicts strong sequence-dependent helix-loop competitions in the pseudoknot stability and the resultant conformational switches between different hairpin and pseudoknot structures. For instance, for the pseudoknot domain of human telomerase RNA, a native-like and a misfolded hairpin intermediates are found to coexist on the (equilibrium) folding pathways, and the interplay between the stabilities of these intermediates causes the conformational switch that may underlie a human telomerase disease

Improvement of RNA secondary structure prediction using RNase H cleavage and randomized oligonucleotides

RNA secondary structure prediction using free energy minimization is one method to gain an approximation of structure. Constraints generated by enzymatic mapping or chemical modification can improve the accuracy of secondary structure prediction. We report a facile method that identifies single-stranded regions in RNA using short, randomized DNA oligonucleotides and RNase H cleavage. These regions are then used as constraints in secondary structure prediction. This method was used to improve the secondary structure prediction of Escherichia coli 5S rRNA. The lowest free energy structure without constraints has only 27% of the base pairs present in the phylogenetic structure. The addition of constraints from RNase H cleavage improves the prediction to 100% of base pairs. The same method was used to generate secondary structure constraints for yeast tRNAPhe, which is accurately predicted in the absence of constraints (95%). Although RNase H mapping does not improve secondary structure prediction, it does eliminate all other suboptimal structures predicted within 10% of the lowest free energy structure. The method is advantageous over other single-stranded nucleases since RNase H is functional in physiological conditions. Moreover, it can be used for any RNA to identify accessible binding sites for oligonucleotides or small molecules

Relativistic corrections in (gamma,N) knockout reactions

We develop a fully relativistic DWIA model for photonuclear reactions using the relativistic mean field theory for the bound state and the Pauli reduction of the scattering state which is calculated from a relativistic optical potential. Results for the 12C(gamma,p) and 16O(gamma,p) differential cross sections and photon asymmetries are displayed in a photon energy range between 60 and 257 MeV, and compared with nonrelativistic DWIA calculations. The effects of the spinor distortion and of the effective momentum approximation for the scattering state are discussed. The sensitivity of the model to different prescriptions for the one-body current operator is investigated. The off-shell ambiguities are large in (gamma,p) calculations, and even larger in (gamma,n) knockout.Comment: LaTeX2e, 18 pages, and 6 figure

Control, Test and Monitoring Software Framework for the ATLAS Level-1 Calorimeter Trigger

The ATLAS first-level calorimeter trigger is a hardware-based system designed to identify high-pT jets, electron/photon and tau candidates and to measure total and missing ET in the ATLAS calorimeters. The complete trigger system consists of over 300 customdesignedVME modules of varying complexity. These modules are based around FPGAs or ASICs with many configurable parameters, both to initialize the system with correct calibrations and timings and to allow flexibility in the trigger algorithms. The control, testing and monitoring of these modules requires a comprehensive, but well-designed and modular, software framework, which we will describe in this paper

Analysis of the initial performance of the ATLAS Level-1 Calorimeter Trigger

The ATLAS first-level calorimeter trigger is a hardware-based system designed to identify high-pT jets, electron/photon and tau candidates and to measure total and missing ET in the calorimeters. The installation of the full system of custom modules, crates and cables was completed in late 2007, but, even before the completion, it was being used as a trigger during ATLAS commissioning and integration. During 2008, the performance of the full system has been tuned during further commissioning and cosmic runs, leading to its use in initial LHC data taking. Results and analysis of the trigger performance in these runs will be presented

Digital signal integrity and stability in the ATLAS Level-1 Calorimeter Trigger

The ATLAS Level-1 calorimeter trigger is a hardware-based system with the goal of identifying high-pT objects and to measure total and missing ET in the ATLAS calorimeters within an overall latency of 2.5 microseconds. This trigger system is composed of the Preprocessor which digitises about 7200 analogue input channels and two digital processors to identify high-pT signatures and to calculate the energy sums. The digital part consists of multi-stage, pipelined custom-built modules. The high demands on connectivity between the initial analogue stage and digital part and between the custom-built modules are presented. Furthermore the techniques to establish timing regimes and verify connectivity and stable operation of these digital links will be described

Testing and calibrating analogue inputs to the ATLAS Level-1 Calorimeter Trigger

The ATLAS Level-1 Calorimeter Trigger is a hardwarebased system which aims to identify objects with high transverse momentum within an overall latency of 2.5 μs. It is composed of a PreProcessor system (PPr) which digitises 7200 analogue input channels, determines the bunch crossing of the interaction, applies a digital noise filter, and provides a fine calibration; and two subsequent digital processors. The PreProcessor system needs various channel dependent parameters to be set in order to provide digital signals which are aligned in time and have proper energy calibration. The different techniques which are used to derive these parameters are described along with the quality tests of the analogue input signals

25 years of African trypanosome research:From description to molecular dissection and new drug discovery

The Molecular Parasitology conference was first held at the Marine Biological laboratory, Woods Hole, USA 25 years ago. Since that first meeting, the conference has evolved and expanded but has remained the showcase for the latest research developments in molecular parasitology. In this perspective, I reflect on the scientific discoveries focussed on African trypanosomes (Trypanosoma brucei spp.) that have occurred since the inaugural MPM meeting and discuss the current and future status of research on these parasites