The RNA World hypothesis describes a period of time during the origins of life in which RNA molecules performed all catalysis and were the only form of information storage. A great deal of evidence has been obtained in support of this hypothesis, however a few key demonstrations are lacking. The first demonstration is of a molecule capable of self-replication that could have plausibly arisen from the prebiotic soup. Previously in the Lehman Laboratory, a 198-nucleotide RNA was discovered that could be fragmented into as many as four pieces ranging from 39 - 63 nucleotides in length. When these pieces were incubated together in a test tube, they re-formed the necessary covalent bonds to regenerate the full-length 198-nucleotide RNA. Furthermore, the full-length RNAs were catalytically active and made copies of themselves from the remaining pieces in solution, providing a model system of self-replication. I was able to remove >10% of the total length of the RNA, which substantially reduced the catalytic activity of the full-length molecule. I discovered several mutations that restored catalytic activity by improved folding and increased catalytic rates using in vitro selection. A subset of these mutations was found to aid in the assembly of the shortened full-length RNA from smaller fragments than were possible in the original system, enhancing the prebiotic relevance of this system. A second demonstration to bolster the RNA World hypothesis would be showing that RNA is capable of harvesting energy from its environment by performing oxidation and reduction reactions. Again using in vitro selection, I have completed five rounds of selection geared towards identifying a ribozyme that reduces benzoic acid to benzaldehyde using Zn2+ and NADH. Results to date suggest the selection is working and it should be continued for another five to ten generations. Finally, I have discovered an RNA sequence that forms knots during transcription, a phenomenon heretofore undocumented in RNA. This new topology has implications for RNA stability by rendering RNA more resistant to hydrolysis, and could impact catalysis through formation of more complex, knotted active sites. Taken together, these findings have improved our understanding of RNA folding and catalysis, and the plausibility of the RNA World.

Thesis (Ph. D.)--University of Chicago, Department of Biochemistry and Molecular Biology, 2001. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI Company.

Not available / text

Mobile group II introns are retroelements that site-specifically insert into DNA target sequences. The group II intron mobility pathway is mediated by a ribonucleoprotein particle (RNP) composed of excised intron RNA and an intron-encoded protein (IEP). The intron lariat inserts at a specific DNA target sequence and is then reverse transcribed by the IEP. Both the intron RNA and IEP are required for DNA target site recognition. I have identified the contact sites within the IEP responsible for recognition of two key positions in the DNA target, T+5 and T-23. IEP recognition of T+5 in the 3'-exon is required for endonuclease cleavage of the bottom-strand of the DNA target site, which generates a primer used for initiation of reverse transcription of the intron. The T+5 base is contacted by G498 in the LtrA DNA-binding domain and nearby residues, particularly K499, potentially bolster this interaction. Recognition of T-23 in the distal 5'-exon is required for initial recognition of the DNA target site by the RNP. The T533 side-chain contacts the T-23 base and the L534 side-chain may also contribute to recognition through hydrophobic interactions with the C5 methyl group. A mutant, L534H, that switches target site specificity to T-23G has been characterized. In order for the RNP to make these and other contacts in the 5'- and 3'-exons simultaneously, the DNA must be bent. I have dissected the role of DNA bending in the intron mobility pathway and found that the DNA is bent at two progressively larger angles as the reaction proceeds. The predominant bend angle at earlier time points places the bottom-strand DNA cleavage site at the protein endonuclease active site. The predominant bend angle of later time points places the cleaved DNA site at the RT domain active site for initiation of reverse transcription of intron cDNA. Finally, in a practical application of group II intron mobility, I have used reprogrammed group II introns ("targetrons") to target two genes in Bacillus subtilis to demonstrate the suitability of targetron technology for gene targeting in the Gram-positive Bacillus genus. / text

Thesis (Ph.D.)--Bowling Green State University, 2008. / Document formatted into pages; contains x, 126 p. : ill. Includes bibliographical references.

The RNA World hypothesis proposes a period of time during the origins of life in which RNA molecules were the only source of both genotypes and phenotypes. Although a vast amount of evidence has been obtained in support of this hypothesis, a few critical demonstrations are lacking. A most crucial one is a demonstration of self-replication of RNA molecule from prebiotic soup. Previously in the Lehman laboratory, it has been demonstrated that a 198-nucleotide molecule derived from the Azoarcus group I intron can self-assemble from up to four fragments of RNA via recombination. Furthermore, the covalent full-length molecules are catalytically active and can make copies of themselves from the remaining pieces in the solution leading to their autocatalytic growth. I was able to demonstrate how this recombination system can overcome different obstacles and evolve to be an efficient replicating system. I discovered the ability of a single RNA fragment to be multifunctional in a single reaction pathway during RNA recombination events that avoids the necessity of multiple genotypes. I also confirmed the capacity of self-replicating ribozymes to form cooperative catalytic cycles and networks that would potentially prevent informational decay. Finally, I have discovered a recycling phenomenon in the RNA recombination system that exploits dynamic covalent chemistry. Recycling provides the earliest replicating system with adequate concentrations of reagents and ability to explore sequence space. Together these findings have improved our understanding of RNA recombination and bolstered the plausibility of the RNA World.

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.