Browsing by Author "Paul Wollenzien, Committee Chair"

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    Prediction and Modeling of the Structure of 16S rRNA
    (2005-11-29) Fanning, Patricia Babin; Paul Agris, Committee Member; Stu Maxwell, Committee Member; Paul Wollenzien, Committee Chair; James Brown, Committee Member
    The ribosome is a complex macromolecule responsible for the translation of genetic information into proteins. Experimental data has provided clear evidence that the ribosome undergoes conformational changes during the process of translation. The size of the ribosome coupled with its dynamic nature has made it difficult to determine the detailed structure of the ribosome using traditional structural analysis techniques (e.g. NMR or crystallography). Recently, crystal structures of the ribosome of several organisms have been determined. The crystal structures are consistent with ~90% of the data obtained experimentally. The areas of the crystal structure of the ribosome which are inconsistent with experimental data are concentrated primarily in the areas of the ribosome known to be actively involved in the process of translation. The computational techniques utilized in this project provide an alternative method for generating structures that are consistent with all the experimental data. In the first phase of the project, MC-SYM, a constraint satisfaction algorithm, was used to generate detailed three-dimensional models for two regions in the RNA portion of the small subunit of the ribosome known to be actively involved in translation: 921- 930/1387-1396 and 1399-1410/1490-1504. The models generated are consistent with the predicted secondary structure, chemical reactivity data, mutagenesis data and crosslinking data. The second phase of the project used statistical methods to identify the highest probability base triples in the small subunit of the ribosome. The most likely candidates for base triples in the ribosome were identified as base triples involving the single stranded nucleotide 121and the 124:237 or the 125:236 base pair. Isomorphic modeling using the algorithm ISOPAIR indicated that a base triple consisting of an interaction between 121 and the 124:237 was the most likely to produce an isomorphic structure for the most frequently occurring sequences for this region. MC-SYM was used then used to construct a model for the 122-127/234-239 base paired region that included a base triple interaction between the single stranded nucleotide 121 and the 124:237 base pair.
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    RNA Dynamics and Interactions Investigated by Photocrosslinking
    (2007-06-19) Huggins, Bruce Wayne; Dennis Brown, Committee Member; Stuart Maxwell, Committee Member; Paul Wollenzien, Committee Chair
    A complete understanding of protein synthesis requires description of the cyclical motions and interactions that occur in ribosomal RNA and transfer RNA during translation. The purpose of the following research was to gain understanding of the nature of these motions and interactions, and to develop new tools to measure how ligands alter the conformational flexibility of RNA during the steps of initiation, elongation and termination. Different tRNA substrates were bound to the E. coli 70S to simulate the arrangement of the tRNA-ribosomal complex before and after peptide bond formation, and different UV-induced 16S rRNA-tRNA photocrosslinks were produced in these complexes, illustrating that the 16S rRNA P-site undergoes local deformations during elongation. A statistical study was undertaken to understand the nature of conformational states in the 30S ribosomal subunit. Using the lists of observed UVB/C- and UVA-s4U-induced crosslinks and the T. thermophilus 30S X-ray crystal structure, frequencies were compared to a number of geometrical parameters demonstrating that crosslink formation requires substantial RNA motions. In addition, the results show that the restricted pattern of crosslink formation in E. coli 16S rRNA is due to the overall rigidity of the 30S subunit outside of the active site. One consequence of these conclusions is that potocrosslinking rates depend on the ease of inter-nucleotide conformational movements. This was exploited in a study that used the temperature response of the rate constant for the UVA-induced photo-crosslink between s4U8 X C13 in E. coli tRNA to determine tRNA geometry and internal energy. The rate constants followed Arrhenius behavior in their dependence on temperature, and this allowed calculation of the activation energy associated with the conformational rearrangement necessary to bring the photoreactive bonds together. The experiments show that changes in the tRNA on ribosomes can be uncovered by photocrosslinking, that RNA mobility occurs by transient conformational changes, and describe a new technique than can quantitatively measure the internal energy associated with these conformational movements.