Interaction of the Bacillus stearothermophilus ribsosomal protein S15 with rRNA

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 1997.Includes bibliographical references (v. 2, 277-305).by Robert T. Batey.Ph.D

Preparation of Isotopically Labeled Ribonucleotides for Multidimensional NMR Spectroscopy of RNA

A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR

Visualising the past – an evaluation of processes and sequences for fingermark recovery from old documents

This study aimed to collect data on the effectiveness of most of the fingermark visualisation reagents currently used on porous surfaces on fingermarks aged for up to 90 years, significantly extending the timescales for which such information exists. A limited subset of the variables associated with processing of old fingermarks was explored, with a focus on the use of 1,8 diazafluoren-9-one (DFO), 1,2-indandione, ninhydrin, and physical developer. These techniques were used in sequence on batches of cheques between 11 and 32 years old, and on documents dating from the 1920s and 1940s. The potential for applying a physical developer enhancement process (blue toning) as the final step in the sequence was also explored. The benefits of using processing sequences on porous items were clearly demonstrated, with all processes in the sequence adding value in terms of additional marks found on the cheques up to 32 years old. In addition, physical developer was found to be capable of developing fingermarks up to 90 years old, whereas the amino acid reagents appear less effective on documents of 70 years and older. An experimental physical developer formulation with reduced environmental impact was found to be as effective as the existing process in these experiments. Blue toning was found to visualise an additional 10-25% of marks, and its wider use after silver-based deposition processes is recommended based on the evidence from this study.Peer reviewedFinal Accepted Versio

hnRNPK recognition of the B motif of Xist and other biological RNAs

Heterogeneous nuclear ribonuclear protein K (hnRNPK) is an abundant RNA-binding protein crucial for a wide variety of biological processes. While its binding preference for multi-cytosine-patch (C-patch) containing RNA is well documented, examination of binding to known cellular targets that contain C-patches reveals an unexpected breadth of binding affinities. Analysis of in-cell crosslinking data reinforces the notion that simple C-patch preference is not fully predictive of hnRNPK localization within transcripts. The individual RNA-binding domains of hnRNPK work together to interact with RNA tightly, with the KH3 domain being neither necessary nor sufficient for binding. Rather, the RG/RGG domain is implicated in providing essential contributions to RNA-binding, but not DNA-binding, affinity. hnRNPK is essential for X chromosome inactivation, where it interacts with Xist RNA specifically through the Xist B-repeat region. We use this interaction with an RNA motif derived from this B-repeat region to determine the RNA-structure dependence of C-patch recognition. While the location preferences of hnRNPK for C-patches are conformationally restricted within the hairpin, these structural constraints are relieved in the absence of RNA secondary structure. Together, these results illustrate how this multi-domain protein's ability to accommodate and yet discriminate between diverse cellular RNAs allows for its broad cellular functions. </section

Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules

Porosity and Structure of Hierarchically Porous Ni/Al2O3 Catalysts for CO2 Methanation

CO2 methanation is often performed on Ni/Al2O3 catalysts, which can suffer from mass transport limitations and, therefore, decreased efficiency. Here we show the application of a hierarchically porous Ni/Al2O3 catalyst for methanation of CO2. The material has a well-defined and connected meso- and macropore structure with a total porosity of 78%. The pore structure was thoroughly studied with conventional methods, i.e., N2 sorption, Hg porosimetry, and He pycnometry, and advanced imaging techniques, i.e., electron tomography and ptychographic X-ray computed tomography. Tomography can quantify the pore system in a manner that is not possible using conventional porosimetry. Macrokinetic simulations were performed based on the measures obtained by porosity analysis. These show the potential benefit of enhanced mass-transfer properties of the hierarchical pore system compared to a pure mesoporous catalyst at industrially relevant conditions. Besides the investigation of the pore system, the catalyst was studied by Rietveld refinement, diffuse reflectance ultraviolet-visible (DRUV/vis) spectroscopy, and H2-temperature programmed reduction (TPR), showing a high reduction temperature required for activation due to structural incorporation of Ni into the transition alumina. The reduced hierarchically porous Ni/Al2O3 catalyst is highly active in CO2 methanation, showing comparable conversion and selectivity for CH4 to an industrial reference catalyst