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The reaction kinetics and three-dimensional architecture of a catalytic RNA

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dc.contributor.advisor David P. Bartel. en_US Bergman, Nicholas H. (Nicholas Henry), 1973- en_US
dc.contributor.other Massachusetts Institute of Technology. Dept. of Biology. en_US 2005-08-23T21:27:56Z 2005-08-23T21:27:56Z 2001 en_US 2001 en_US
dc.description Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001. en_US
dc.description Includes bibliographical references. en_US
dc.description.abstract The Class I ligase ribozyme was isolated previously from random sequences based on its ability to promote a reaction similar to a single step in RNA polymerization: attack of a primer 3'-hydroxyl on a 5'-triphosphate, with formation of a new 3'-5' bond and release of pyrophosphate. Derivatives have been shown to catalyze general primer extension reactions, making the ligase a useful paradigm for RNA self-replication and RNA polymerase biochemistry as well as RNA catalysis in general. In order to establish the ligase as a model system, we have characterized both the reaction and tertiary architecture of the ribozyme. The reaction kinetics of both multiple- and single-turnover ligation were examined, and from these data minimal kinetic frameworks were constructed. These frameworks provide a basis for the interpretation of future mechanistic work, and suggest strategies by which individual steps in the ligation reaction might be targeted for future improvement. In order to test whether the chemical step of Class I ligation could be further optimized, an in vitro selection was performed under conditions that specifically isolated chemistry. Selected variants had a slightly improved chemical step, and substantially improved Mg++-dependence, such that at 0.5 mM Mg++ a composite improved ligase was 50-fold faster than the parent ribozyme. The tertiary architecture of the ligase was examined using hydroxyl radical probing, which provided a measure of the solvent accessibility at each position in the RNA backbone. In collaboration with another group, these data were used to model the tertiary architecture of the ligase in three dimensions. Finally, the predictive value of the model was.tested and confirmed by photocrosslinking experiments. en_US
dc.description.statementofresponsibility by Nicholas H. Bergman. en_US
dc.format.extent 188, [7] leaves en_US
dc.format.extent 12774940 bytes
dc.format.extent 12774697 bytes
dc.format.mimetype application/pdf
dc.format.mimetype application/pdf
dc.language.iso eng en_US
dc.publisher Massachusetts Institute of Technology en_US
dc.rights M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. en_US
dc.subject Biology. en_US
dc.title The reaction kinetics and three-dimensional architecture of a catalytic RNA en_US
dc.title.alternative Reaction kinetics and 3D architecture of a catalytic ribonucleic acid en_US
dc.type Thesis en_US Ph.D. en_US
dc.contributor.department Massachusetts Institute of Technology. Dept. of Biology. en_US
dc.identifier.oclc 49264557 en_US

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