Dynamic Kinetic Selection (DKS) suggests that kinetic, rather than thermodynamic, stability will dictate the composition of a replicating population of biomolecules. Here, the results obtained from a series of five related reactions involving gradually increasing percentages of randomly-mutated substrate fragments to generate variants of full-length Azoarcus group I intron through an autocatalytic self-assembly reaction involving a series of recombination events, showed DKS as a driving factor in dictating the population composition of full-length product assembled from substrates that had fewer positions available to randomization.
In trying to elucidate a plausible scheme for the origins of complex biomolecules on the prebiotic Earth, the suggestion that networks comprised of interacting molecules were more likely to evolve into biomolecules capable of obtaining and sustaining characteristics attributed to living molecules has gained traction within the past few years. Of specific interest is the catalytic efficacy of ribozymes whose genotypes require that they interact with molecules of the same genotype (selfish systems) to be effective catalysts versus those that are more effective when accomplishing catalysis by cooperating with ribozymes of a different genotype (cooperative systems). Here, the Azoarcus I ribozyme was used to compare these two types of system. Both systems were shown to robustly produce full-length product. Two different methods of introducing random mutations into substrate fragments for the reactions described in this thesis were employed. The differences in the preparation methods for the substrates was not expected to have an impact on the nature of the full-length product. However, there was no correlation between the positions that tended to be more tolerant of accepting random mutations between the products arising from the two preparation methods. One preparation method yielded full-length ribozymes more consistent with the secondary structure of the wild-type ribozyme and followed substitution patterns found in in vivo nucleic acid substitutions, whereas the other method provided full-length ribozymes that tolerated mutations that would be expected to greatly affect the secondary structure of the ribozyme and those positions tended to mutate evenly to any of the three possible alternative nucleobases.
Point mutations introduced into ribozyme substrate fragments may have a deleterious, neutral, or beneficial effect, depending on their impact on the catalytic capability of the molecule vis-á-vis the effect, if any, the change has to the secondary and tertiary structure of the ribozyme. In this dissertation, the results of two series of point mutation reactions are addressed. The first set showed a point mutation to have a deleterious effect, whereas concerted mutations did not significantly affect activity of the ribozyme. The second series of reactions involved point mutations at a position that had previously been determined to be highly tolerant of random mutations. Results suggested that substitutions at this position had a minimal impact on ribozyme activity.
|Advisor:||Lehman, Niles E.|
|Commitee:||Benight, Albert S., Duffield, Deborah A., Reichow, Stephen L.|
|School:||Portland State University|
|School Location:||United States -- Oregon|
|Source:||DAI-B 80/08(E), Dissertation Abstracts International|
|Keywords:||Dynamic kinetic selection, Molecular evolution, Ribozyme, Self-assembly|
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